Hydrogen fluoride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

toxicity to reproduction

Remarks:

other: male reproductive organ physiology

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Published study, no guideline followed; no assessment of fluoride levels in the diet or drinking water.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Assessment of male reproductive organs following subacute oral exposure to sodium fluoride.

GLP compliance:

no

Remarks:

: published literature study

Limit test:

no

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

Healthy adult male Swiss mice weighing 20-30g were maintained on standard chow and water ad libitum. They were housed in an air-conditioned animal house at a temperature of 26±2oC and exposed to 12-14 daylight hours. Mice were aged 6-8 weeks old at the beginning of the experiment.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Sodium fluoride, dissolved in doubly distilled water, was administered to mice orally using a feeding tube attached to a hypodermic syringe.

Details on mating procedure:

Not examined.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No information

Duration of treatment / exposure:

30 days

Frequency of treatment:

Daily

Details on study schedule:

Not applicable; mice were not mated.

Remarks:

Doses / Concentrations:
10 or 20 mg/kg bw
Basis:
nominal in water
administered by gavage

No. of animals per sex per dose:

40 mice per group, 5 groups

Control animals:

yes, concurrent no treatment

Details on study design:

One group of mice remained untreated to act as a control. Two groups of mice were administered the test substance orally for 30 days at either 10 or 20 mg/kg bw, and subject to autopsy on day 31. A further two groups of mice was administered 10mg/kg bw for 30 days, and allowed to recover for 30 or 60 days prior to autopsy. Mice were divided into weight-matched treatment groups. Doses were chosen based on previous reports and the LD50 of fluoride in mice.

Positive control:

Not examined

Parental animals: Observations and examinations:

No observations were reported prior to sacrifice.

Oestrous cyclicity (parental animals):

Not applicable

Sperm parameters (parental animals):

See below

Litter observations:

Not applicable

Postmortem examinations (parental animals):

Mice were sacrificed and the following organs removed, fixed in Bouin's fixative, embedded in paraffin wax and sectioned: testis, caput and cauda epididymides, vas deferens, prostate and seminal vesicles. Sections were stained by haematoxylin-eosin staining. Sections were utilised for histocytometric measurements.

Postmortem examinations (offspring):

Not applicable

Statistics:

A minimum of 30 replicates of each histocytometric parameter was obtained and analysed using the Student's t-test. The level of significance was determined using Fisher and Yates table.

Reproductive indices:

Not applicable

Offspring viability indices:

Not applicable

Clinical signs:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

not examined

The histoarchitecture of the testis was altered after treatment as compared to controls. The seminiferous tubule diameter decreased with NaF treatment at 20mg/kg bw. The germinal epithelium was disorganised with denudation of cells in the lumen, which was devoid of sperm. The germinal epithelial cell height was significantly reduced, however diameters of Leydig cells and their nuclei remained unaltered. Withdrawal of treatment resulted in recovery in the histology of the testis compared to the treated group. The diameter of tubules in the caput epididymis was increased in treated animals, but not significantly. The secretory epithelium showed nuclea pyknosis and no sperm were observed in the lumen. The cauda epididymis of treated mice revealed larger tubules with extensive denudation of cells in the lumen, which waws devoid of sperm. The epithelial cell height was significantly decreased. A marked recovery was observed in the histology of the epididymides after withdrawal of treatment. The epithelium of the vas deferens showed nuclear pyknosis. The diameter of the vas deferens and the thickness of the muscle coat were significantly less than in control animals. Again, withdrawal of treatment resulted in recovery. No changes were observed in the seminal vesicle or prostate gland.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Not applicable - not examined.

Reproductive effects observed:

not specified

Sodium fluoride treatment of male mice resulted in alterations in the histoarchitecture of the testis.

Conclusions:

Sodium fluoride treatment of male mice resulted in alterations in the histoarchitecture of the testis. Sperm was completely absent from the tubular lumen. The effects seen after 30 days administration were reversible.

Executive summary:

Sodium fluoride was administered orally by gavage to groups of male mice at doses of 0, 10 or 20 mg/kg bw/d. Mice were sacrificed and the testes subject to histological and histocytochemical evaluation. Sodium fluoride treatment of male mice resulted in alterations in the histoarchitecture of the testis: severe disorganisation and denudation of germinal epithelial cells of seminiferous tubules; sperm was completely absent from the tubular lumen; a reduction in epithelial cell height, nuclear pkynosis, denudation of cells and absence of sperm occurred in the cauda epididymis; the vas deferens epithelium showed nuclear pyknosis, with no sperm in the lumen. The effects seen after 30 days administration were reversible. The levels of fluoride in the diet and drinking water were not reported in this study, therefore signifcantly reducing its reliability and value.

Reference 2

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Published FDA study

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 reprodcutive toxicity study, however this paper focuses on investigations of the effects on spermatogenesis in male rats following administration over two generations. Additional findings are reported in other papers by the same authors.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were male and female Charles River CD VAF+ (Sprague-Dawley) rats, aged 22 days on arrival and quarantined for approximately 1 week. Individuals were identified by ear tags. Rats were housed under standard controlled temperature (67-74oF), 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. and is the same formulation used in the NTP study (1990).

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 determiined 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

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:

Doses / Concentrations:
0, 25, 100, 175 or 250ppm
Basis:
nominal in water

No. of animals per sex per dose:

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

Positive control:

Not examined

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.

Litter observations:

No information.

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.

Reproductive indices:

Not examined.

Offspring viability indices:

Not examined.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Dose descriptor:

NOAEL

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

See above (parental animals) for results.
In addition to the above; liver weights on the 100 and 250ppm groups were significantly lower than controls. This was considered a random occurrence by the authors because no dose-related effects were observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 ppm (nominal)

Sex:

male

Basis for effect level:

other: Spermatogenesis and endocrine function

Reproductive effects observed:

not specified

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.

Reference 3

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Published FDA study

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

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The male rats were from the larger two-generation study reproduction study (Sprando et al 1997). They were male and female Charles River CD VAF + Sprague-Dawley rats, aged 22 days on arrival and quarantined for approximately 1 week. Individuals were identified by ear tags. Rats were housed under standard controlled temperature (67-74oF), 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. and is the same formulation used in the NTP study (1990).

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 concentration 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

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, 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:

Doses / Concentrations:
0, 25, 100, 175 or 250ppm
Basis:
nominal in water

No. of animals per sex per dose:

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

Positive control:

Not examined

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

Litter observations:

No information.

Postmortem examinations (parental animals):

Reported in Sprando et al 1997

Postmortem examinations (offspring):

Some results are reported in Sprando et al 1997.

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.

Clinical signs:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

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

Remarks on result:

other: P0 not evaluated, only F1-generation male rats evaluated.

Dose descriptor:

NOAEC

Effect level:

> 250 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

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

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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 250 ppm (nominal)

Sex:

male

Basis for effect level:

other: No adverse effects on testis structure or spermatogenesis at any dose level.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Sodium fluoride exposure during prenatal development stages (through parental exposure), and for 14-weeks at weaning had no adverse affect on spermatogenesis in F1-gen male rats at doses up to 250 ppm.

Reproductive effects observed:

not specified

The significant reduction in the absolute volume of the testicular capsule in the 100ppm 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.

Conclusions:

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

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.

Reference 4

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published non-guideline study

Qualifier:

according to guideline

Guideline:

other: WHO protocol MB-50 (1983).

Principles of method if other than guideline:

The effects of orally administered sodium fluoride on sperm. A fertility test was carried out according to WHO protocol MB-50 (1983).

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: albino Charles Foster

Sex:

male

Details on test animals or test system and environmental conditions:

Healthy, intact, mature, pathogen-free male albino rats, weighing 225-230 g, obtained from National Institute of Occupational Health. The animals were acclimatised for 7 days. They were maintained under standard conditions, standatd diet and water were provided ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Sodium fluoride was dissolved in distilled water and administered using a feeding tube attached to a hypodermic syringe.

Details on mating procedure:

Oestrus or prooestrus untreated females were cohabited with treated males (2 females to 1 male) on the 50th day of treatment according to the WHO protocol. Vaginal smears were taken the next morning to check for the presence of sperm. If sperm was found it was assumed that the female would be pregnant, and was termed day 0 of pregnancy.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No information available.

Duration of treatment / exposure:

50 ± 2 days

Frequency of treatment:

Daily

Details on study schedule:

NaF was administered to male rats for 50 days. On day 50 they were mated to untreated females. The males were sacrificed after mating.

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 males per group

Control animals:

yes, concurrent no treatment

Details on study design:

An additional treatment group was included; these rats were given NaF for 50 days, then allowed to recover for another 70 days.

Positive control:

Not examined.

Parental animals: Observations and examinations:

No information available.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Sperm motility and count of the cauda epididymal sperm. Hyaluronidase activity, acrosin activity, differential staining of spermatozoa by silver nitrate to study sperm acrosomal integrity.

Litter observations:

Not examined.

Postmortem examinations (parental animals):

The males were killed after completion of treatment and the cauda epididymal spermatozoa obtained.

Postmortem examinations (offspring):

Not determined.

Statistics:

ANOVA followed by Students-Newman-Keuls test

Reproductive indices:

Fertility rate - no information is given regarding the calculation of the index

Offspring viability indices:

Not determined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

NaF treatment caused a significant inhibition of cauda epididymal sperm motility compared to untreated controls. Withdrawal of treatment for 70 days resulted in a significant recovery. The cauda epididymal sperm declined significantly in treated rats. A trend toward recovery occurred after 70 days.
Fertility was inhibited by treatment, and was partially restored following the 70 day recovery period.
Treatment also caused a significant reduction in hyaluronidase activity and total acrosin activity. Sperm in the NaF treated group exhibited loss of acrosome and deflagellation. Poor recovery was seen in the 70 day recovery period.

Dose descriptor:

dose level:

Effect level:

10 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Effects on sperm count and motility

Reproductive effects observed:

not specified

NaF treatment caused a significant inhibition of cauda epididymal sperm motility compared to untreated controls. Withdrawal of treatment for 70 days resulted in a significant recovery. The cauda epididymal sperm declined significantly in treated rats. A trend toward recovery occurred after 70 days.

Fertility was inhibited by treatment, and was partially restored following the 70 day recovery period.

Treatment also caused a significant reduction in hyaluronidase activity and total acrosin activity. Sperm in the NaF treated group exhibited loss of acrosome and deflagellation. Poor recovery was seen in the 70 day recovery period.

Conclusions:

Sodium fluoride caused reversible changes to sperm in the rat following oral administration

Executive summary:

Male rats were adiministered sodium fluoride by gavage for 50 days at a dose of 10 mg/kg bw. One group of rats were allowed a 70 recovery period after the treatment period. Sperm count, motility and fertility were adversely effected following treatment (compared to untreated controls). Acrosomal hyaluronidase and acrosin activity were reduced in treated rats. Sperm morphology was also adversely affected in treated rats. The 70 day recovery period resulted in partial recovery of the above parameters. It should be noted that the authors do not report the fluoride content on the diet and water, and the measures of fertility are poorly reported.

Reference 5

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Published study, no guideline followed, levels of fluoride in the diet and drinking water are not reported.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Assessment of male reproductive organs following subchronic oral exposure to sodium fluoride.

GLP compliance:

no

Remarks:

: published study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Male albino CD rats, 60-75 days old, weighing 200-250g. Rats were maintained in temperature and light controlled condition (14h light : 10h dark). Water was provided ad libitum. Animal assignment to treatment groups was random. Standard pelleted rat diet was used as feed.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Sodium fluoride was added to standard pelleted rat diet.

Details on mating procedure:

Treated and control rats were mated with untreated females. Rats were cohabited for 4 days at a ratio of two females to one male. Vaginal smears were taken every morning to check for the presence of sperm. Females were kept for confirmation of pregnancy until parturition.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males were exposed to sodium fluoride in the diet for 60 days

Frequency of treatment:

Continuous (ad libitum)

Details on study schedule:

Males were exposed for 60 days, and either mated to females for 4 day, or sacrificed on day 60.

Remarks:

Doses / Concentrations:
0, 100 or 200 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

Fifteen males per dose; 10 males per dose mated to 20 untreated females. 5 males per dose were sacrificed for examination of reproductive organs.

Control animals:

yes, concurrent no treatment

Details on study design:

No further information

Positive control:

None.

Parental animals: Observations and examinations:

Blood samples were collected by cardiac puncture from 5 rats in each group. Serum was separated for testosterone radioimmunoassay.

Oestrous cyclicity (parental animals):

Not examined, females were not exposed to the substance

Sperm parameters (parental animals):

Spermatogenesis was evaluated by measuring the inner diameter of the seminiferous tubules, and the thickness of the peritubular membranes. The percentage of seminiferous tubules containing spermatozoa attached to Sertoli cells was counted.

Litter observations:

The number of newborns and average litter size was calculated.

Postmortem examinations (parental animals):

5 males were sacrificed for examination of testes after 60 days exposure; testes were removed, trimmed, fixed in 10% neutral formalin, embedded in paraffin, sectioned and stained with haematoxylin and eosin.

Postmortem examinations (offspring):

Not examined

Statistics:

The Student's t-test was used to compare experimental and control values

Reproductive indices:

Number of pregnant females

Offspring viability indices:

Not examined

Clinical signs:

no effects observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Control male rats showed a normal mating response and mated females gave birth to normal litters.

Treated rats showed less interest towards females. The number of pregnant females and number of newborns was significantly reduced in both exposed groups compared to controls. There was also a reduction in litter size in the exposed groups, but this was not significant. Exposed rats displayed a reduction in the diameter of seminiferous tubules compared to controls. The thickness of the peritubular membranes was significantly increased in the 200ppm rats, and these rats also exhibited a significant reduction in the percentage of seminiferous tubules containing spermatozoa. Serum testosterone levels were significantly decreased in the 200 ppm rats, and non-significantly decreased in the 100 ppm rats.

Dose descriptor:

NOAEL

Effect level:

< 100 ppm

Sex:

male

Basis for effect level:

other: Adverse effects on male fertility are reported at dose levels of 100 and 200 ppm sodium fluoride.

Clinical signs:

not examined

Mortality / viability:

not specified

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Reproductive effects observed:

not specified

The effect of fluoride intake on fertility

Treatment Group	No. Males	No. Females	No. Pregnant Females	No. Newborns	Average Litter Size ± SEM
Control	10	20	18 (90%)	197	10.94 ± 0.941
100 ppm NaF	10	20	16 (80%)	137	8.56 ± 0.741
200 ppm NaF	10	20	10 (50%*)	84*	8.40 ± 0.498

Average litter size was calculated for pregnant females in each group

* 200ppm vs. control P<0.05

 

The effect of fluoride intake on seminiferous tubule diameter and peritubular thicknes

Treatment Group	Mean tubular diameter (µm)	Peritubular membrane thickness (µm)
Control	296.1 ± 1.835	2.463 ± 0.051
100 ppm NaF	288.6 ± 2.042**	42.5 ± 3.862
200 ppm NaF	275.7 ± 2.512**	33.0 ± 2.390*

Values are mean ± SEM

* 200ppm vs. control P<0.05

** 100ppm and 200ppm vs. control P<0.01

Conclusions:

The results of this study indicate an adverse effect of sodium fluoride on male fertility.

Executive summary:

The effects on reproductive performance of sodium fluoride, added to the diet of male rats for 60 days at concentrations of 100 ppm and 200 ppm, were investigated. Exposure resulted in an appparent reduction in fertility, based on number of pregnant females and number of newborns, and changes in diameter of the seminiferous tubules. Mean serum testosterone levels of 5 rats showed a non-significant decrease with increasing dose. Estimations of fluoride intake (i.e. feed intake), and fluoride content of the diet, were not made, thereby significantly limiting the value and reliability of this study.

Reference 6

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Published study of non-standard design; no reporting of background fluoride levels in the diet or drinking water.

Qualifier:

according to guideline

Guideline:

other: fertility test conducted according to WHO Protocol MB50 (1983)

Principles of method if other than guideline:

Assessment of fertility in male rats

GLP compliance:

no

Remarks:

: published literature study

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

Healthy adult colony bred male rats (Rattus norvegicus) weighing between 200-250g were maintained on standard chow and water was provided ad libitum. Four rats per cage were housed in an air conditioned animal house at 26±2oC and exposed to 12-14 hours of daylight. The rats were two months old at the start of the experiment.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Sodium fluoride was dissolved in double distilled water.

Details on mating procedure:

Treated male rats were mated with untreated females

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

30 days

Frequency of treatment:

Daily

Details on study schedule:

Rats were administered the test subtance orally by gavage for 30 days, then sacrificed.

Remarks:

Doses / Concentrations:
5 or 10 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

No information

Control animals:

yes, concurrent no treatment

Details on study design:

No further information

Positive control:

Not examined

Parental animals: Observations and examinations:

Body weights, there is not clear information but it appears body weights were taken at the sacrifice.

Oestrous cyclicity (parental animals):

Not applicable.

Sperm parameters (parental animals):

The cauda epididymidal sperm suspension was prepared in normal saline. The percentage motility and count of cauda epididymal spermatozoa of control and treated rats were determined by the method of Prasad et al (1972) and expressed as percentage and millions/ml respectively. The fertility test was carried out according to the WHO protocol MB50 (1983).

Litter observations:

Not applicable - rats were not mated

Postmortem examinations (parental animals):

Testis, caput and cauda epididymides, vas deferens, prostate, and seminal vesicles were dissected out, blotted and weighed. The following data were also collected: level of serum testosterone, the activity of succinate dehydrogenase (SDH), the amount of cholesterol in the testis, the activity of adenosine triphosphatase (ATPase), sialic acid concentrations in caput and cauda epididymides, the activity of acid phosphatase, the protein levels in the ventral prostate gland, and the concentration of fructose in the seminal vesicle and vas deferens.

Postmortem examinations (offspring):

Not applicable - rats were not mated

Statistics:

A minimum of 8 replicated of each parameer were done and the data analysed by the students t-test. P values less than 0.05 were considered significant.

Reproductive indices:

Not applicable

Offspring viability indices:

Not applicable

Clinical signs:

not examined

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

not examined

There was no difference in body weight between the control and low dose groups, the high dose group showed a small statistically significant decrease compared to controls. The cauda epididymal sperm density and motility decreased significantly. Serum testosterone levels were not significantly affected. SDH activity in the testis showed a dose dependent decrease. Testicular cholesterol content remained unaffected. ATPase activity was reduced in both caput and cauda epididymides and the reduction was greater in cauda epididymides. The concentration of sialic acid in both cauda and caput epididymides was significantly decreased. The ventral prostate gland acid phosphatase activity showed no change in the low dose rats, whereas a significant increase was observed in the high dose rats. Ventral prostate protein was not significantly altered by low dose sodium fluoride, but it was enhanced by the high dose. Seminal vesicle fructose levels were not affected. The vas deferens fructose level showed a dose dependent decrease as compared to control rats. Table 1 displays the body weight, fetility and sperm data.

Dose descriptor:

LOAEL

Effect level:

< 5 mg/kg bw/day (nominal)

Sex:

male

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Not applicable - not examined.

Reproductive effects observed:

not specified

Body weights, sperm parameters, fertility and serum testosterone levels in NaF treated rats

Parameter	Control	Sodium fluoride treated
		5 mg/kg bw	10 mg/kg bw
Body weight (g)	260±15.04	273±33.89	237±7.8
Sperm motility (%)	62±1.41	40.26±0.96	29.13±6.29*
Sperm count (millions/ml)	58±2.16	32.51±2.1*	15.98±3.89*
Fertility rate (%)	95	25*	16*
Serum Testosterone (ng/ml)	3.4±0.18	3.07±.0.49	2.93±0.56

Values are mean ± SE

* P<0.001

Conclusions:

Sodium fluoride administration reduced fertility and had various spermatological effects in male rats.

Executive summary:

Sodium fluoride was administered orally to adult male rats daily for 30 days at either 5 or 10 mg/kg bw. Body weights were reduced in the high dose group compared to untreated controls. Succinate dehydrogenase activity in the testis was inhibited. Adenosine triphosphatase and sialic acid levels in epididymides were also suppressed, particularly in the cauda epididymidis. Sperm motility and count were decreased, leading to a significant decline in fertility by fluoride treatment. There were no data regarding the fluoride content of the diet or feed/water intake, thereby significantly limiting the value of this study.

Reference 7

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: FDA GLP study published in peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD CRL:CD-BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

CD-BR VAF rats, obtained from Charles River Laboratories, USA. Males ad females weighed 51-75g on receipt. They were acclimatised for 1 week, and identified individually by ear tags. Rats were fed low-fluoride NIH-07 diet (7.95ppm fluoride, Ziegler Bros., USA). Single animals were housed in stainless steel cages suspended in racks. Pregnant females and females with litters were housed in polycarbonate tubs with Sani-Chips as bedding. Light in the animal room was provided on a 12 h light/dark cycle. The average temperature was 71-73oF, and average humidity was 35-67%.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

NaF was administered in drinking water. Weight/volume NaF solutions were prepared in water obtained by filtering house-distilled water through a Hydro Pico pure water system. The concentration of fluoride in the filtered water was <0.2ppm.

Details on mating procedure:

Rats in the F0 generation were mated on a 1:1 basis. Females that failed to mate after 1 week were remated to a different male within the same treatment group. Each female was allowed up to 3 weeks for mating. Cohabitation began at approximately 16.30h on each mating day. Mating was confirmed by the presence of sperm in the vaginal lavage. Females that mated were presumed pregnant. Rats were randomly selected from the resultant F1 generation for mating following the same procedure as in the F0 generation. Litter mates were not mated. The day mating was confirmed was designated as gestation day 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

10 weeks.

Frequency of treatment:

Daily.

Details on study schedule:

F0 parental animals were exposed to NaF for 10 weeks. They were then mated randomly within treatment groups. At gestation day 20 8 females per group were subject to caesarean section and examination, these results were reported elsewhere. The remaining females were allowed to litter and wean their pups to postnatal day 21 (the day of birth was designated postnatal day 0). On postnatal day 4 litters were culled to 10 pups by random procedure.

On postnatal day 21 36 F1 males and 36 F1 females per group were randomly selected for mating (no more than 2 of each sex per litter). After 10 weeks NaF exposure they were allowed up to 3 weeks to mate. At gestation day 20, caesarean sections were performed on the pregnant females (the results are discussed elsewhere).

Remarks:

Doses / Concentrations:
0, 25, 100, 175, or 250 ppm
Basis:
nominal in water

No. of animals per sex per dose:

F0 generation: 48 rats per sex per dose
F1 generation: 36 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were based on the National Toxicology Program (1990) chronic two year study, plus an additional higher dose (250ppm) based on a developmental toxicity study conducted previously by the authors (Collins et al 1995).

Positive control:

Not examined

Parental animals: Observations and examinations:

Body weights were recorded during the 10 week exposure period.
All animals were examined individually on a dialy basis for clinical signs and mortality.
Feed and water consumption were measured during the 10 week exposure period.

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Not examined as part of this study (reported elsewhere).

Litter observations:

The number of still born pups was noted. On postnatal days 0, 4, 7, 14 and 21 each pup was observed, sexed and weighed. Litters were culled to 10 pups per litter on postnatal day 4. The incidence of runts was calculated on postnatal days 0, 4, 7, 14 and 21 (the weight of individual pups was compared to the average of litter averages per sex, any animal weighing less tha 70% of the grand mean weight was termed a runt).

Postmortem examinations (parental animals):

Ten males and females from each group (F0 adults, F1 weanlings and F0 adults) were evaluated for histopathological effects. All gross lesions were recorded, animals were weighed, and organ weights were determined for the: thymus, heart, kidneys, adrenal glands, brain, liver, testes, epididymides, prostate, seminal vesicle, ovaries and spleen. Histopathology was performed on the following tissues for all animals: heart, aorta, spleen, thymus, lungs, liver, kidney, pituitary, adrenal glands, thyroid and parathyroid, trachea, oesophagus, stomach, duodenum, pancreasm jejunum, ileum, cecum, colon, testes, ovaries, urinary bladder, epididymides, semival vesicle, prostate, uterus, cervix, vagina, eyes with optic nerves, mammary gland, sternum with marrow, brain, spinal cord, and all gross lesions. In addition, the following tissues were evaluated for animals in the control and 250ppm groups: salivary gland, tongue, mesenteric lymph node, rectum, intraorbital lacrimal glands, psoas muscle, skin, skull, Harderian glands, teeth, nasal turbinates, vertebral column, right femur with marrow and right sciatic nerve. All tissues were observed from sections stained in haematoxylin and eosin.

Postmortem examinations (offspring):

See above.

Statistics:

Clinical signs were analysed by Fishers Exact test. Feed consumption, and reproductive data were analysed using ANOVA. LSD tests were used to compare controls with each treated group. Body weight and weight gain, organ weights and ravid weight were compared between groups using ANCOVA and LSD tests.
Fluid consumption was contraindicated by several animals that played with the water tubes. Outliers were removed from the statistical analysis using the Grubb's test.
P values of less than or equal to 0.05 were considered significant.

Reproductive indices:

Mating index - (no. sperm positive/no. exposed to mating) x100
Fertility index #1 - (no. produced litter/no. sperm positive) x100
Fertility index #2 - (no. produced litter/no. exposed to mating) x100

Offspring viability indices:

Number of live births, runts, growth.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

No dose-related clinical effects were observed in either males or females.

There was a significant decrease in overall feed consumption by F0 males in the 250ppm group. Rats in the 175 and 250ppm groups drank significantly less than controls. This decreased consumption is attributed to decreased palatability. Weight gain of males and females showed a significant negative linear regression, however only the individual weight gain of the 250ppm males was statistically significantly less than controls.

Female mating indices were over 90% in all groups. Female fertility indices were decreased slightly in the 250 ppm group, but not significantly. Average time to mating was less in treated groups than in the controls but the differences were not dose related.

Mean water consumption per pregnant female was decreased in the 250ppm group during the entire period of gestation.
There were no effects on organ weights or organ to body weight ratios.
There was an increase in the development of prominent growth lines in the upper incisors of all rats that received 250ppm. Hyperkeratosis of hte limiting ridge of the stomach was diagnosed in a small number of rats from the 100, 175 and 250ppm groups.

Dose descriptor:

NOAEL

Effect level:

250 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: No effects on reproduction were seen at the highest dose level

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

No dose-related clinical effects were observed in either males or females.

Oveall mean feed consumption of F1 females showed a significant negative linear regression for days 0-70 although none of the values were significantly less than controls. F1 males in the treated groups ate less than the control group but the decreases were neither dose related nor significant. Rats in the 175 and 250ppm groups drank significantly less than controls. F1 males in the 100ppm group drank significantly less than the control group. This decreased consumption is attributed to decreased palatability.
Mating indices of females were over 90%. The fertility indices of the females in thhe 25 and 250ppm were slightly less than controls, but not significantly and probably due to random variation.
All the mating indices of the F1 males were less than those of the F0 males, but there was no dose-related decrease.
Mean fluid consumption per pregnant female was decreased in the 250ppm group during the entire period of gestation. Fluid consumption was significantly decreased in 17 5 ppm group females during the entire period of gestation.
Survival of F1 offspring to postnatal day 21 showed no dose-related effects. Growth and development were similar in all groups, and female and male runts were randomly distributed among the control and treatment groups.
There were no effects on organ weights or organ to body weight ratios.
There was an increase in the development of prominent growth lines in the upper incisors of all rats that received 250ppm. Hyperkeratosis of hte limiting ridge of the stomach was diagnosed in a small number of rats from the 250ppm group.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: No effects on reproduction were seen at the highest dose level

Reproductive effects observed:

not specified

NaF consumption by F0 females ranged from 3.5-27.3 mg NaF/kg bw/d at 25 -250 ppm respectively, and consumption in F1 females was 3.8 -28.0 mg NaF/kg bw/d. Consumption in the F0 males was 2.8 to 23.1 mg NaF/kg/d, and in the F1 males was 3.0 -24.1 mg NaF/kg bw/d.

Conclusions:

Sodium fluoride administered in the drinking water for 10 weeks at dose levels up to 250ppm had no adverse effects on reproduction in rats.

Executive summary:

The effects of sodium fluoride ingestion at 0, 25, 100, 175 or 250 ppm in drinking water was measured in rats over 3 generations.

Reproduction was not affected by NaF administration, and offspring viability also remained unaffected. The decreased fluid consumption noted in high dose groups was attributed to decreased palatability. Mating, fertility and survival indices were not affected. Sodium fluoride caused an increase in the incidence of whitening of tooth enamel, in a dose-related manner from males and females in the 100, 175 and 250ppm groups. There was an increase in the development of prominant growth lines in the upper incisors of F0 and F1 adult rats and F1 weanlings. The reproductive NOAEL in rats was therefore 250 ppm.

Reference 8

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Published study of non-standard design, limited reporting and no assessment of fluoride levels in the diet or drinking water.

Qualifier:

according to guideline

Guideline:

other: WHO Protocol MB50

Principles of method if other than guideline:

Assessment of male fertility following subacute oral exposure to test substance

GLP compliance:

no

Remarks:

: published literature study

Limit test:

no

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

Healthy adult Swiss mice weighing 20-30g. The mice were maintained on standard chow, water was given ad libitum. The mice were housed in an air conditioned animal house at a temperatire of 26±2oC and exposed to 12-14 daylight hours. The mice were divided into a control group and four treatment groups.

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

Sodium fluoride was adminstered orally for 30 days.

Details on mating procedure:

Normal cycling females were cohabited with treated males the day after the 30 day exposure period, or at the end of the withdrawal period (one or two months). Females were placed with males at a ratio of 2:1. Vaginal smears were checked every morning the presence of sperm, if sperm was found the female was separated, allowed to remain on a normal diet for 16 days then autopsied.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

30 days treatment. Mice were then sacrificed after treatment termination, 1 month after treatment termination, or 2 months after treatment termination.

Frequency of treatment:

Daily.

Details on study schedule:

Males were treated for 30 consecutive days. Mice were mated and sacrificed the day after treatment termination, 1 month after treatment termination, or 2 months after treatment termination. Females were maintained on normal diet for 16 days of pregnancy prior to sacrifice.

Remarks:

Doses / Concentrations:
10 or 20 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

20 male mice per dose, mated 2:1 with untreated females.

Control animals:

yes, concurrent no treatment

Details on study design:

No further information

Positive control:

Not examined

Parental animals: Observations and examinations:

No observations were made prior to sacrifice.

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Cauda epididymidis sperm motility and sperm count were evaluated. The acrosomal integrity of the sperm from the cauda epididymis was studied using the modified silver nitrate technique.

Litter observations:

Not examined

Postmortem examinations (parental animals):

Males were autopsied for assessment of cauda epididymdis. Females were autopsied and the uterus evaluated for number of implantation sites and corpora lutea, although these results are not reported.

Postmortem examinations (offspring):

Not examined

Statistics:

No information

Reproductive indices:

No information

Offspring viability indices:

Not examined.

Clinical signs:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

not examined

The cauda epididymis sperm motility was significantly decreased in the two treatment groups. Withdrawal of treatment for 2 months resulted in almost complete recovery.
The cauda epididymal sperm count was decreased in treated mice compared to controls. Recovery was noted after 2 months without treatment.
Scanning electron microscopy showed that treated mice cauda epididymis spermatozoa had head midpiece and tail abnormalities compared to controls. Deflagellated spermatozoa were also observed. Silver nitrate staining of cauda epididymal sperm showed clear differences between control and treated mice.

Dose descriptor:

NOAEL

Effect level:

< 10 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: Reduced fertility was seen in males at 10 and 20 mg/kg bw/d

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: Reduced fertility was seen in males at 10 and 20 mg/kg bw/d

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings:

not examined

No information

Reproductive effects observed:

not specified

Thirty days of oral sodium fluoride administration induced a loss of fertility as a result of damage to male reproductive organs. However this damage was found to be reversible.

Conclusions:

The findings of this study are consistent with the results of other studies by the same authors.

Executive summary:

Thirty days of oral sodium fluoride administration at doses of 10 and 20 mg/kg bw induced a loss of fertility in mice. Sperm count and motility were significantly reduced. Large numbers of spermatazoa were deflagellated and possessed abnormalites. A significant recovery in sperm count, motility and fertility rate occurred after 2 months. This study does not specify the method of oral exposure, or give any reference to fluoride contents in the diet alongside body weights and food and water consumption. The methods state that pregnant female mice were autopsied on gestation day 16 for evaluation of uterine contents; the results of this examination are not presented. The absence of information on background levels of fluoride in the diet and drinking water significantly limits the value of the study.

Reference 9

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published peer reviewed study

Principles of method if other than guideline:

The study is a 2-generation reproduction study in mice exposed to the test substance in the drinking water.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

Swiss Webster

Sex:

female

Details on test animals or test system and environmental conditions:

The animals were weanling random bred Swiss Webster albino female mice, obtained from Simonsen Laboratories, USA. Mice were fed a low fluoride diet (the composition is provided below) ad libitum. The diet provided 0.1 to 0.3 ppm fluoride, on a dry weight basis. Deionised water (with or without NaF added) was provided ad libitum. The mice were housed in polypropylene cages with stainless steel wire lids, and wood shavings were provided as litter and nesting material.
Breeding groups consisted of 4 females to 1 male per cage.

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

Female mice were fed a low fluoride diet (0.1-0.3 ppm fluoride), plus drinking water containing 0, 50, 100 or 200 ppm fluoride as sodium fluoride for 25 weeks.

Details on mating procedure:

First generation: Breeding groups of 4 females and 1 male were established when the females reached 8 weeks of age, and males were housed constantly with females to allow postpartum mating. Males were a minimum of 10 weeks old at first mating, and were transferred between groups every 6 weeks. Litters were reduced to 6 pups at birth, and all pups were removed from the mothers after 5 days. This time period was chosen as the minimum necessary to distinguish between a postpartum conception and conception following cesstion of lactation. Litter production was assessed up to a maximum of 4 litters during the 25 week period.
Second generation: reproduction was assessed in mice from the low fluoride diet control group and the 50 ppm group. Female pups from litter 4 (and some from litter 3) were raised to weaning at 21 days and fed the same fluoride intake as their dams. The mating procedure was identical to that used for the first generation.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

The diet was analysed for fluoride content, no further information is provided.

Duration of treatment / exposure:

25 weeks

Frequency of treatment:

Daily in drinking water

Details on study schedule:

Females were exposed to fluoride from weanling. Mating began once they reached 8 weeks of age, litters were removed from the mothers after 5 days to allow re-mating. The authors state that with a gestation period of 3 weeks, healthy mice are capable of producing up to 8 litters in the 25 week exposure period. Males were transferred between mating groups every 6 weeks to ensure there were no effects of fluoride intake.
Second generation mice in the 0 ppm and 50 ppm groups from litters 3 and 4 were raised to weaning and then exposed to the same level of fluoride as their dams. The mating procedure then followed that used for the first generation.

Remarks:

Doses / Concentrations:
0, 50, 100 or 200 ppm
Basis:
nominal in water
an additional 0.1-0.3 ppm fluoride was present in the diet

No. of animals per sex per dose:

First generation females: 0 ppm n = 58; 50 ppm n = 55; 100 ppm n = 50 and 200 ppm n = 50. There were appproximately 54 males matched to the females.
Second generation females: 0 ppm n = 38 and 50 ppm n = 44. There were approximately 21 males matched to the females.

Control animals:

yes

Details on study design:

First generation female mice were dividied randomly into treatment groups. Second generation females were taken mainly from litter 4 with some from litter 3, and were exposed to the same level of fluoride as their dams.

Positive control:

Not examined.

Parental animals: Observations and examinations:

Mortality was recorded. Body weights were recorded weekly from weaning until 8 weeks of age. The age of the dams at the delivery of their first litter was recorded, along with the time interval between their litters, and the number of the litters per dam.

Oestrous cyclicity (parental animals):

Not determined directly, although the time interval between litters gives an indication of the oestrus cycle.

Sperm parameters (parental animals):

Not examined.

Litter observations:

The number of pups per litter was recorded. The weights at birth and 5 days of age was recorded for randomly selected pups (100 newborn and 50 5 day old pups per fluoride group) were recorded for the 0 ppm and 50 ppm groups.

Postmortem examinations (parental animals):

Humeri obtained at 3, 8 and 33 weeks of age were ashed and analysed for fluoride using the fluoride ion-specific electrode.

Postmortem examinations (offspring):

Humeri obtained at 3 and 33 weeks of age were ashed and analysed for fluoride using the fluoride ion-specific electrode.

Statistics:

Overall litter production was compared between the 0 ppm and 50 ppm groups for each of the 4 litters (both generations) using a comparison of binomial distributions. Infertility proportions for different litters were compared within the 0 ppm group only using the chi-square test. Infertility proportion: proportion of mice failing to produce a litter. Age at delivery of first litter and time intervals between litters were compaed between the 0 and 50 ppm groups using the students t-test. The frequency of postpartum conception was compared in the 0 and 50 ppm groups for litters 2, 3, and 4 of both generations by a comparison of binomial distributions.

Reproductive indices:

Overall litter production: % mice in each group producing a given number litters.
Frequency of postpartum conception: a positive postpartum conception was recorded if a litter was produced within 24 days of the previous litter, taking into account a possible increase in the gestation period in response to the combined stresses of pregnancy and lactation.

Offspring viability indices:

Not examined.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Mice in the 0 ppm group and 50 ppm group gained weight at an identical rate, and at 8 weeks weighed (mean±SEM) 25.1±0.33 g and 25.2±0.43 g, respectively. Mice in the 100 ppm and 200 ppm groups gained weight more slowly, this was apparent after 2 weeks in the 100 ppm mice and after 1 week in the 200 ppm mice. Mice in the 200 ppm group failed to gain weight after 5 weeks of age, and 50% had died by 8 weeks of age. All mice in the 200 ppm had died by 20 weeks of age. Insignificant losses occurred in mice fed the lower fluoride levels.

Overall litter production: No litters were produced by mice in the 200 ppm group. Only 9 litters were born over a 10 week period in the 100 ppm group, and 6 of these litters were still-born or eaten at birth. Both these groups were excluded from further analysis. In the 50 ppm group, 96% of mice produced 4 litters (not significantly lower than 100%). There was a significant reduction in litter production with successive litters in the 0 ppm group; 100% produced 1 litter whilst < 50% produced 4 litters.

Infertility proportions: The proportions were low in the 50 ppm group indicating that reproduction was not significantly impaired in this group. The proportion was zero in the 0 ppm group for litter 1, but increased in subsequent litters. An average of 18.3% mice became infertile after each litter in the 0 ppm group.

Age at first litter: Mice in the 0 ppm and 50 ppm groups were approximately 13 weeks old when they delivered their first litter.

Intervals between litters: There were no differences in interval between the 0 ppm and 50 ppm groups, or between generations. The interval was approximately 4-5 weeks.

Frequency of postpartum conceptions: In the 50 ppm group, 50 - 61.2% of eligible litters resulted from postpartum conceptions. The 0 ppm group achieved a comparable percentage except for litter 4 where there was a decrease to 23.1%.

Fluoride concentrations in humeri: The concentrations averaged 0.017±0.001% of the ash in weanling mice. When the mice were placed into the 0 ppm group, the fluoride concentration declined by 8 weeks of age and remained low throughout the breeding period. In the 50 ppm group, the fluoride concentration increased prior to breeding, and was 70-80 times greater than that of the 0 ppm group.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified Generation not specified (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Second generation mice in the 0 ppm and 50 ppm groups gained weight at a slightly greater rate than in generation 1 and at 8 weeks weighed (mean±SEM) 27.9±0.33 g and 28.3±0.29 g, respectively.

Maternal fluoride intake did not affect pup body weight at birth or 5 days of age in the 0 ppm and 50 ppm groups. Litter sizes were comparable for both groups, averaging approximately 9 pups over both generations.

Overall litter production: In the 50 ppm group, 90% of mice produced 4 litters (not significantly lower than 100%). There was a significant reduction in litter production with successive litters in the 0 ppm group; 81.5% produced 1 litter whilst < 50% produced 4 litters.

Age at first litter: Mice in the 50 ppm group were approximately 13 weeks old when they delivered their first litter. Mice in the 0 ppm group did not deliver their first litter until 16 weeks of age.

Intervals between litters: There were no differences in interval between the 0 ppm and 50 ppm groups, or between generations. The interval was approximately 4-5 weeks.

Fluoride concentrations in humeri: The fluoride concentrations in the 0 ppm group were comparable to those in generation 1. Weanling mice of the 50 ppm group had a higher fluoride concentration (0.072±0.01% of the ash) than in generation 1, but the levels at 33 weeks of age were similar in both generations.

Reproductive effects observed:

not specified

A high degree of fluoride toxicity was seen at 100 ppm, as evidenced by reduced growth rate and impaired reproduction, and at 200 ppm as evidenced by the high mortality rate. Data from these groups were excluded from analysis. The results suggest that 50 ppm fluoride in drinking water (approximately 7.5 mg F^-/kg bw/day) is more adequate to maintain reproductive capacity than the low fluoride control diet without fluoride supplemented drinking water.

Conclusions:

Fluoride toxicity was evident at concentrations of 100 ppm and 200 ppm. Fluoride did not appear to impair reproduction at 50 ppm.

Executive summary:

A 2 generation study was conducted with sodium fluoride. Female Swiss-Webster mice were fed a low fluoride diet (0.1 -0.3 ppm fluoride) plus drinking water containing 0, 50, 100 or 200 ppm fluoride for 25 weeks from weaning. Females were mated with untreated males. A high degree of fluoride toxicity was seen at 100 ppm, as evidenced by reduced growth rate and impaired reproduction, and at 200 ppm evidenced by a high mortality rate. Also, in the 100 ppm only 9 litters were born over a 10 week period. In the control group a progressive decline in litter production with successive litters occurred in both generations. The results suggest that 50 ppm fluoride in drinking water (approximately 7.5 mg F^-/kg bw/day) is more adequate to maintain reproductive capacity than the low fluoride control diet without fluoride supplemented drinking water.

Reference 10

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Published peer reviewed study. The study reliability of the study may have been affected by the occurrence of an infection in some of the study animals.

Principles of method if other than guideline:

The study was a three-generation study investigating the effects of dietary sodium fluoride on reproduction.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

Swiss Webster

Sex:

female

Details on test animals or test system and environmental conditions:

The animals were weanling female Webster mice weighing 10±3 g, obtained from from Rolfsmeyer Company, USA. The mice were randomly assigned to treatment groups. Mice were housed in groups of 4 or 5 in stainless steel cages. No litter material was used, food and deionised water were provided ad libitum. Pregnant females were housed singly with paper as nesting material.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

First generation mice were fed a low fluoride diet (composition below), or a low fluoride diet supplemented with 2 ppm fluoride. Second and third generation females were fed the same diet as their dams, except in the second generation a third group was supplemented with 100 ppm fluoride.

Details on mating procedure:

First generation: Breeding groups were established by introducing 1 male to each cage when the females reached 8 weeks of age, and was continued for 25 weeks. Females were removed from group housing when they were confirmed pregnant, as indicated by an increase in body weight. After 5 days pups were removed and the mothers returned to the breeding cages. Each female was allowed to have a maximum of four litters during the 25 week period. Breeding males were exchanged between cages every 4 weeks to eliminate any effect of fluoride intake.
Second and third generations: The second and third generations were selected from fourth litter pups of the previous generation. They were raised to weaning by the dams, following which they were fed the same diets as their dams. The mating procedure was identical to that used in the first generation, except overnight breeding was used. An additional treatment group was included in the second generation, these mice were fed the low fluoride diet supplemented with 100 ppm fluoride (the group was composed of equal numbers of offspring from the 0 ppm and 2 ppm groups).
Litter size was reduced to 6 at birth.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

An analysis of the basal diet was conducted, according to the same method used to determine levels of fluoride in tissue (see below).

Duration of treatment / exposure:

25 weeks

Frequency of treatment:

Daily in diet

Details on study schedule:

Females were exposed to fluoride from weanling. Mating began once they reached 8 weeks of age, litters were removed from the mothers after 5 days to allow re-mating. Males were transferred between mating groups every 5 weeks to ensure there were no effects of fluoride intake.
Second generation mice from litter 4 were raised to weaning and then exposed to the same level of fluoride as their dams, with the creation of an additional 100 ppm group. Third generation females were selected from the fourth litters of the second generation. The mating procedure then followed that used for the first generation.

Remarks:

Doses / Concentrations:
0, 2 or 100 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

There were 29-35 females per group.

Control animals:

yes

Details on study design:

First generation mice were randomly distributed to treatment groups. Second generation mice were exposed to the same diet as their dams, except for the additional group exposed to 100 ppm, which comprised an equal number of offspring from the 0 ppm and 2 ppm groups. Third generation mice were exposed to the same diet as their dams. Litter size was reduced to 6 at birth.

Positive control:

Not examined.

Parental animals: Observations and examinations:

Body weights were recorded at least weekly.

Oestrous cyclicity (parental animals):

Not determined directly, although the number of litters produced within the 25 week period can give an indication of cyclicity.

Sperm parameters (parental animals):

Not examined.

Litter observations:

The total number and weight of pups per litter were recorded. Pups were reweighed at 5 days old (prior to termination). The number of stillborn pups was recorded.

Postmortem examinations (parental animals):

Blood samples were obtained under anaesthesia, by cardiac puncture immediately prior to termination. Hematocrit was determined.
The liver, kidneys and femur were removed and used for fluoride copper and iron analyses.

Postmortem examinations (offspring):

See above. Also plasma samples were collected for fluoride determination in the third generation.

Statistics:

A pooled t-test procedure was used.

Reproductive indices:

Not determined.

Offspring viability indices:

Not determined.

Clinical signs:

not examined

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

No difference in growth rate was determined between groups in the first generation, and mice in both groups weighed approximately 29 g at 8 weeks of age.

There were no effects on reproduction in the first generation. There was no effect on fluoride treatment on hematocrit levels in breeding females in any generation.

Dose descriptor:

NOEL

Effect level:

100 ppm (nominal)

Sex:

female

Basis for effect level:

other: No effects of fluoride treatment on reproduction

Remarks on result:

other: Generation not specified (migrated information)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Mice in the second and third generations grew at a slightly greater rate than the first generation, with no differences between treatment groups. The weight at 8 weeks of age for all groups in the two generations ranged from 31.5 to 34.2 g.

Fertility was slightly decreased in litter 4 in the second generation mice fed 0 ppm. As this effect was not seen in the other generations and did not follow a dose-response relationship it was not thought related to fluoride treatment.
In the third generation, reproductive rate for fourth litter production was decreased in all treatment groupa, and subsequent autopsies determined the breeding males in all groups were carrying a kidney infection.

There were no effects on the size of litters, the average number of pups per litter ranged from 10 to 13. There was no influence of generation, litter number within the generation or fluoride level. The birth weight of the pups in all groups was about 1.6 g, and when they were re-weighed at 5 days old there was no effect on weight gain in the second and third generation pups. The weight gain of the first generation pups was significantly higher than the second and third generations (2.6 g compared to 1.7 g).

The incidence of stillbirth was extremely variable within any litter of any generation, ranging from 3% to 21%.

A significant decrease in femur fluoride content was seen in mice fed 0 ppm compared to 2 ppm or 100 ppm in all 3 generations. The fluoride concentration in bone continued to increase as the dietary fluoride content or ingestion time increased.

Plasma fluoride levels in the third generation were higher in the 100 ppm group than the other two groups. The concentration increased as the exposure time increased.

Hematocrit values tended to fluctuate in offspring (samples taken at birth and at 5 days of age), although there were no significant differences.

Reproductive effects observed:

not specified

No effects of fluoride toxicity were reported. There were no apparent effects of fluoride administration on reproduction across the 3 generations, however the study may have been affected by the presence of a kidney infection in the breeding males.

Conclusions:

No compound-related effects on reproduction were observed. The presence of a kidney infection may have affected the reliability of the study.

Executive summary:

A 3 -generation study was conducted with female mice. The first generation were exposed to 0 ppm and 2 ppm fluoride (as NaF), in a low fluoride diet, and mated with untreated males. Second and third generation females received 0, 2 and 100 ppm fluoride in the low fluoride diet. The low fluoride diet contained less than 0.5 ppm fluoride. No signs of fluoride toxicity were reported. No compound-related effects on reproduction were observed. The presence of a kidney infection may have affected the reliability of the study.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

NaF (read-across analogue to HF), Klimisch 1 GLP studies to OECD 416 (Collins, 2001, Sprando 1997, 1998). Remaining studies are Klimisch 2 & 3 non-GLP supporting studies to no guideline or a non-OECD specified guideline.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Published studies

Araibi et al (1989) report adverse effects on the fertility of male rats administered sodium fluoride in the diet at concentrations of 100 and 200 ppm. Exposure resulted in a reduction in successful matings and reduced litter size; findings were associated with a reduction in seminiferous tubule diameter and a thickened peritubular membrane. The numbers of tubules containing spermatozoa were decreased and serum testosterone levels were also reduced. Chinoy & Sequeira (1989) report alterations in the histoarchitecture of the testes in mice gavaged with sodium fluoride at dose levels of 10 and 20 mg/kg bw/d for 30 days. Findings were characterised by severe disorganisation and denudation of germinal epithelial cells of the seminiferous tubules, absence of sperm from the tubular lumen, reduced in epithelial cell height, nuclear pkynosis, denudation of cells and absence of sperm occurred in the cauda epididymis. The effects seen after 30 days administration were reversible. Chinoy et al (1992) report reduced fertility in male rats administered sodium fluoride by gavage at dose levels of 5 and 10 mg/kg bw for 30 days. Findings were accompanied by reduced sperm count and motility and various biochemical changes in the testes.

The results of these studies are consistent, however their value and reliability is significantly compromised by the absence of any information on the fluoride levels in diet and/or drinking water. The actual levels of fluoride exposure cannot be accurately assessed. It is also notable that the findings of these published investigative studies of non-standard design contrast with the total absence of reproductive toxicity at comparable dose levels in the FDA studies reported below.

Messer et al (1973) investigated the reproductive toxicity of sodium fluoride in a two-generation study in which female mice were administered the test material in the drinking water at dose levels of 0, 50, 100 or 200 ppm. A progressive decline in litter production was seen in the control group. All females administered 200 ppm fluoride died over the study period; only a small number of litters were produced at the 100 ppm. It is suggested that a level of 50 ppm sodium fluoride (equivalent to approximately 7.5 mg/kg bw.d fluoride) is required to maintain reproductive capacity in female mice. In a 3 -generation mouse study (Tao & Suttie, 1976), no effects of fluoride on reproduction were seen. The study is of limited value, however the authors suggest that the effects of fluoride seen in the study of Messeret al (1973) was due to the influence of fluoride on teh absorption of iron from a low iron diet.

FDA studies

The effects of sodium fluoride administration on spermatogenesis in rats were investigated in a two-generation study (Sprando et al, 1997). In contrast to the previous studies, no effects were observed on reproductive organ weights, sperm parameters or biochemical parameters at dose levels of up to 250 ppm (drinking water). Additional detailed investigations by the same authors did not reveal any effects on spermatogenesis in F1 males (Sprando et al, 1998). No effects on reproduction were seen at the highest dose level of 250 ppm in a guideline-comparable two-generation rat study (Collins et al, 2001). In a further FDA study designed primarily to assess the potential effects of fluoride on spermatogenesis (as indicated in various published studies), Sprando et al (1996) demonstrated that injection of sodium fluoride into the rat testis was without effect on spermatogenesis.

In contrast to the other studies which report effects of fluoride on male fertility and spermatogenesis, no effects were observed in the FDA studies following extensive investigation. The two-generation FDA study is of standard design and is comprehensively reported, and it is notable in these studies that the contribution of diet and drinking water to the total fluoride intake was assessed. The EU RAR for HF also considers the data available for the reproductive toxicity of NaF and concludes that the FDA studies are key, for reasons of design, reporting and control of fluoride levels. The EU RAR concludes that the NOAEL for reproductive toxicity is 250 ppm NaF, which corresponds to approximately 10 mg/kg bw/d fluoride. The absence of any apparent effects on the reproductive organs in chronic toxicity and carcinogenicity studies is also notable.

Short description of key information:
No studies with HF are available. However a number of studies of various designs are available with the read-across substance NaF, including high quality studies performed by the US FDA.

Effects on developmental toxicity

Description of key information

No studies with HF are available.  However a number of studies of various designs are available with the read-across substance NaF, including high quality studies performed by the US NTP and FDA.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

The animals were Caesarean-derived, viral anitbody-free (CD-CRL:CD-BR, VAF+) rats obtained from Charles River Laboratories, MA, USA. On arrival the males weighed 351-375g, and the females weighed 175-200g. During the study, rats were housed in stainless steel cages suspended in stainless steel racks. Light was provided on a 12 hour light/dark cycle (light 07.30-19.30hr). The temperature was 17.8-25.6oC (64-78oF). Humidity ranged from 15-73%; the lowest humidity value (15%) was recorded once during the first week of the study. During the remainder of the study the range of low humidity values was 24-42%. During mating rats were housed in groups of 2 females and 1 male. Female rats were fed low-fluoride NIH-07 diet (7.95ppm fluoride). The diet was identical to that used in the NTP (1990) study obtained from Ziegler Bros. Inc., PA, USA.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Weight/volume NaF solutions were prepared in Aqua Cool Ultra Pure water (Ionics, Inc., MA, USA). Test solutions were presented to rats as drinking water available ad libitum. Only female rats were exposed to NaF drinking water (males were used as sires only).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No information available

Details on mating procedure:

Rats were cohabited from approximately 16.30hr on each mating day, until the following morning. The ratio of females to males was 2:1. On each morning after cohabitation, the females were removed from the mating cages and individually smeared for the presence of sperm in the vaginal lavage. The females that were confirmed as having mated were presumed pregnant and proceeded onto the study.

Duration of treatment / exposure:

20 days (from gestation days 0-20).

Frequency of treatment:

Daily

Duration of test:

From mating until gestation day 20.

No. of animals per sex per dose:

The numbers of females in each dose group (in ascending dose order): 35, 35, 35, 36, 37 and 37.

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were selected on the basis of doses used in the NTP carcinogenicity study (1990), with an additional lower and higher dose to increase the range. Once mating was confirmed, female rats were assigned to treatment groups by stratified random procedure.

Maternal examinations:

Fluid consumption was measured every 3 days, feed consumption was measured on gestation days 7, 14 and 20. Body weights were recorded at 3 day intervals during gestation and on day 20 prior to sacrifice. Behavioural signs and clinical toxicity were recorded. The numbers of corpora lutea were counted and recorded.

Ovaries and uterine content:

Caesarean sections were performed on gestation day 20. Each uterus was examined in situ for the presence and position of resorption sites, implantation sites, and live or dead foetuses. Deciduomas were called early deaths, and implantation sites with placentas and with complete but non-viable foetuses that were of subnormal size, that showed retarded development, or that were in a macerated condition, were classed as late deaths.

Fetal examinations:

Each viable foetus was weighed, sexed, measured for crown-rump length, and examined under magnification for the presence of external abnormalities. Viable foetuses were alternately evaluated for skeletal abnormalities using Alzarin Red S stain or for soft tissue abnormalities following serial sections.

Statistics:

ANOVA and two-tailed LSD test.

Indices:

Average percentage early + late deaths/litter

Historical control data:

No information

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was no dose-related behavioural changes or clinical signs. Water consumption was significantly reduced at 175 and 200pm, and feed consumption was significantly reduced at 250 ppm, body weights reflected feed consumption trends; significant decreases in body weight gain were seen in 250ppm females on days 0-3 and 6-9, and overall on days 0-20. The mean number of implants per litter was significantly reduced in the 250 ppm was significantly decreased, however findings correspond with a lower number of corpora lutea in this group.

Dose descriptor:

NOAEL

Effect level:

175 ppm (nominal)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

250 ppm (nominal)

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
A significant increase was seen in the average number of foetuses with 3 or more skeletal variations in the 250ppm group, and the numbers of litters with foetuses with 3 or more skeletal variations was also increased in this group but not significantly so.

Dose descriptor:

NOAEL

Effect level:

250 ppm (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Water consumption by females was lower at dose levels of 175 and 200 ppm. This resulted in a lower than expected NaF consumption. Pregnancy rate was over 90% in all groups. A significantly lower number of corpora lutea was seen in the dams of the 250 ppm group. Implantation efficiency (% corpora lutea that implanted) was more than 90% in all groups except the 25 ppm group, although the small decrease was not significant. The occurrence of in utero deaths was similar in the control and treated groups. The mean number of male foetuses per litter was significantly decreased in the 175 ppm group compared to the control, but was not dose related therefore considered to be random.

Foetal growth was not affected by NaF, even in the high dose groups with dams exhibiting reduced food and water consumption. Male control foetuses weighed 4.0 g, and crown-rump length was 4.1cm; male foetuses from treated groups weighed 3.9 -4.1g and crown-rump length was 4.0 -4.1cm. Female control foetuses weighed 3.8g and crown-rump length was 4.0 cm; female foetuses from treated groups weighed 3.7 -3.8g and crown-rump length was 3.9 -4.0cm.

Conclusions:

No evidence of developmental toxicity was seen in this study.

Executive summary:

The developmental toxicity of sodium fluoride was determined in rats. Mated females were exposed to sodium fluoride in the drinking water at concentrations of 0, 25, 100, 175 and 250 ppm on gestation days 0 -20. Caesarean sections were performed on gestation day 20 and foetuses were examined. Sodium fluoride was not teratogenic at any dose tested. There was no effect on the development of specific bones including sternebrae. Foetal growth was not affected by sodium fluoride, even in dams exhibiting significantly decreased food and water consumption (250ppm; decreased feed and water consumption, 175ppm decreased water consumption). A significant increase was seen in the average number of foetuses with three or more skeletal variations in the 250 ppm group, however the number of affected litters was not significantly increased. There was no dose related effect on sodium fluoride on the incidence of soft tissue variations.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP study, only abstract available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Female New Zealand White Rabbits were fed standard laboratory chow ad libitum. Water (control and treated) was provided ad libitum.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Sodium fluoride was administered to rats in the drinking water, provided ad libitum.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

The method detection limit was used to determine the level of NaF present in the control water, and this value was used to calculate the drinking water doses. The amount of F present in the standard diet was also determined.

Details on mating procedure:

Rabbits were mated, and dosing began on gestation day 6. No further information is available.

Duration of treatment / exposure:

Treatment ran from gestation day 6 to gestation day 19.

Frequency of treatment:

Daily

Duration of test:

30 days, beginning on gestation day 0.

No. of animals per sex per dose:

26 female rabbits per dose

Control animals:

yes, concurrent vehicle

Details on study design:

No further information.

Maternal examinations:

Animals were observed daily for clinical signs of toxicity. Food, water, and body weights were recorded for the animals in each group on gestation day 0 and every two days thereafter through gestation day 30. Blood samples were collected from 5 animals per group per replicate on gestation day 20; serum was delivered to the sponsor for determination of fluoride concentration. All animals were killed on gestation day 30 and examined for maternal body and organ weights, implant status, foetal weight, sex, and morphological development.

Ovaries and uterine content:

Uterine contents were examined - implant status, foetal weight, sex and morphological development were recorded.

Fetal examinations:

Foetuses were examined for external, visceral or skeletal malformations, in addition to foetal body weights and sex.

Statistics:

No further information

Indices:

No further information

Historical control data:

No further information

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal toxicity occurred. Pregnancy rates were 84%, 87%, 78%, and 83% in the control to high exposure groups, respectively. Maternal body weight change for the animals receiving 400 ppm NaF was significantly lower than that of control animals for the period from gestation day 6 to 8 (14 grams average weight gain for controls vs. 112 grams weight loss for the 400 ppm group); this difference probably resulted from significantly decreased food and water consumption during the same period. Maternal body weight change was significantly increased from gestation day 10 to 12 (22 grams average weight gain for controls vs. 71 grams weight gain for the 400 ppm group), but did not differ among groups for the treatment period as a whole, indicating that animals in the 400 ppm group recovered from the weight change effects observed during the first few days of exposure to NaF in the drinking water. Maternal water consumption (g/kg/day) during exposure was significantly decreased in the animals exposed to 400 ppm NaF. Post-exposure water consumption was normal in these animals indicating the probability of dereased palatability of the 400 ppm solution. Maternal food consumption was decreased compared to control during the first four days of treatment (g/day on gestation day 6 to 8 and 8 to 10; g/kg/day on gestation day 6 to 8), but was normal thereafter. No clear clinical signs of toxicity were observed. Necropsy of the maternal animals revealed no effects on kidney or liver weights.

Dose descriptor:

NOAEL

Effect level:

200 ppm (nominal)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

400 ppm (nominal)

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Exposure did not affect the frequency of post-implantation loss, mean foetal body weight per litter, or external, visceral, or skeletal malformations.

Dose descriptor:

NOAEL

Effect level:

400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on fetal development of rabbits given sodium fluoride in drinking water up to 400 ppm that resulted in decreased maternal water intake.

Abnormalities:

not specified

Developmental effects observed:

not specified

Drinking water contained less than 0.6 ppm of sodium fluoride (the detectable limit). Water intake provided approximately 84%, 91 % and 95% of the total F consumed for the low through high concentration groups in this study. Determination of serum fluoride levels, in 7-8 pregnant animals per group, revealed levels of 0.06 ± 0.04, 0.24 ± 0.10, 0.39 ± 0.14, and 0.70 ± 0.33 ppm at the end of the exposure period for the control through high dose groups, respectively.

Conclusions:

There was evidence of minimal maternal toxicity but no evidence of developmental toxicity with levels of sodium fluoride in drinking water as high as 400 ppm (resulting in an average exposure of 29 mg/kg bw/d) although the palatabillity of a 400 ppm sodium fluoride solution apparently reduced water consumption.

Executive summary:

Pregnant New Zealand White rabbits were exposed to sodium fluoride in their drinking water at concentrations of 0, 100, 200 or 400 ppm daily between gestation days 6 and 19. There was evidence of minimal maternal toxicity but no definitive evidence of developmental toxicity with levels of sodium fluoride in drinking water as high as 400ppm (resulting in an average exposure of 29 mg/kg bw/d) although the palatabillity of a 400 ppm sodium fluoride solution apparently reduced water consumption. This study established a NOAEL for maternal toxicity at 200 ppm NaF in drinking water (approximately 18 mg/kg bw/d) and a NOAEL for developmental toxicity of 400ppm NaF in drinking water (approximately 29 mg/kg bw/d) administered to pregnant NZW rabbits during organogenesis.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP study: abstract available

Qualifier:

according to guideline

Guideline:

other: NTP protocol

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Developmental toxicity study

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Sprague-Dawley CD rats were fed standard laboratory chow ad libitum. Water (control and treated) was provided ad libitum.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Sodium fluoride was administered to rats in the drinking water, provided ad libitum.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

The method detection limit was used to determine the level of NaF present in the control water, and this value was used to calculate the drinking water doses. The amount of F present in the standard diet was also determined.

Details on mating procedure:

No further information is available; assumed pregnant females were dosed

Duration of treatment / exposure:

Treatment from gestation day 6 to gestation day 15.

Frequency of treatment:

Daily

Duration of test:

Animals were treated on Day 6-15 of gestation and sacrificed

No. of animals per sex per dose:

26 female rats per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

No further information

Maternal examinations:

Animals were observed daily for clinical signs of toxicity. Food and water intakes and body weights were recorded on gestation days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. All animals were sacrificed on gestation day 20 and examined for maternal body and organ weights, implant status, foetal weight, sex and morphological development. An additional 10 mated animals per groups were subjected to the same experimental regimen but sacrificed on gestation day 16 for blood collection for determination of serum fluoride concentration.

Ovaries and uterine content:

Uterine contents were examined - implant status, foetal weight, sex and morphological development were recorded.

Fetal examinations:

Foetuses were examined for external, visceral or skeletal malformations, in addition to foetal body weights and sex.

Statistics:

No further information

Indices:

No further information

Historical control data:

No further information

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal lethality occurred at any dose. Maternal weight gain was significantly reduced at 300ppm during the first 2 days of exposure (gestation days 6-8), and a trend toward decreased weight gain was noted for the treatment period as a whole. Maternal food intake was significantly decreased (compared to controls) in the 300ppm group between gestation days 8-10. Water consumption was significantly decreased during exposure in the 300ppm group. No other differences were noted. At necropsy there were no effects on kidney or liver weights.

Dose descriptor:

NOAEL

Effect level:

150 ppm (nominal)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

300 ppm (nominal)

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
NaF exposure did not significantly affect the frequency of post-implantation loss, mean fetal body weight per litter, or external, visceral, or skeletal malformations.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NaF exposure did not significantly affect mean fetal body weight per litter, or external, visceral, or skeletal malformations. As no adverse effects were observed a NOAEL of 300 ppm was established for developmental toxicity (27 mg NaF/kg bw/d) following NaF administered in drinking water to pregnant CD rats during organogenesis.

Abnormalities:

not specified

Developmental effects observed:

not specified

Control water fluoride levels were <0.6 ppm NaF.

Food contained an average of 12.4 ppm F (11.6 -13.4 ppm F).

The calculated doses from drinking water were 7, 18 and 27 mg NaF/kg bw/d (3, 8 and 12 mg F/kg bw/d) for the low, intermediate and high-dose groups respectively. Intake from food added approximately 2 mg NaF/kg bw/d (1 mg F/kg bw/d) to the intake for each group.

Determination of serum fluoride levels in the 10 animals per group terminated on 16 revealed mean levels of 0.007 ± 0.002, 0.035 ± 0.040, 0.039 ± 0.039, and 0.187 ± 0.076F at the end of the exposure period.

Conclusions:

Sodium fluoride in drinking water was not maternally toxic up to doses of 300ppm, although decreased water consumption was seen as a result of poor palatability at this dose. There was no evidence of developmental toxicity in this study.

Executive summary:

Pregnant Sprague-Dawley CD rats were exposed to sodium fluoride in their drinking water at concentrations of 0, 50, 150 or 300 ppm daily between gestation days 6 and 15. Maternal weight gain was significantly reduced at 300 ppm during the first two days of exposure (days 6 to 16). Maternal water consumption (grams/kg/day) during exposure was significantly decreased in the animals exposed to 300ppm NaF. Post-exposure water consumption was normal in these animals indicating the probability of decreased palatability of the 300ppm solution. Necropsy of the maternal animals revealed no effects on kidney or liver weights. NaF exposure did not significantly affect the frequency of post-implantation loss, mean fetal body weight per litter, or external, visceral, or skeletal malformations.

This study established a NOAEL for maternal toxicity of 150 ppm (18 mg NaF/kg bw/d) and a NOAEL of 300 ppm for developmental toxicity (27 mg NaF/kg bw/d) administered in drinking water to pregnant CD rats during organogenesis.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

14 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

All studies were performed using Sodium Fluoride (as a read-across surrogate analogue) to Hydrogen Fluoride.
Klimisch 2 (NTP, 1994) and Klimisch 1 (Collins et al, 1995) (GLP not specified) studies equivalent / similar to OECD 414.
Klimisch 2 (NTP, 1993) (GLP, Not specified), study equivalent / similar to OECD 414 (Collins et al , 1995).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a rat developmental toxicity study (NTP, 1994; Heindel et al, 1996), maternal toxicity (transiently reduced bodyweight gain) was apparent at the highest dose level of 300 ppm sodium fluoride (in drinking water), equivalent to 13 mg/kg bw/d fluoride. No evidence of developmental toxicity was seen at this dose level. No clear evidence of developmental toxicity was seen in an FDA rat study (Collins et al, 1995) at dose levels of up to 250 ppm sodium fluoride in drinking water (equivalent to 12.3 mg/kg bw/d fluoride). Maternal toxicity in this study was limited to reduced food intake at the highest dose level. No evidence of developmental toxicity was seen in a rabbit study (NTP, 1993; Heindel et al, 1996) at dose levels of up to 400 ppm sodium fluoride (equivalent to 14 mg/kg bw/d fluoride from all sources).

Justification for classification or non-classification

Reliable studies do not indicate any developmental toxicity or reproductive toxicity of fluoride. No classification is therefore proposed.