Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published FDA study. Information on actual received dose missing.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
The study design is comparable to a standard two-generation reproductive toxicity study, however this paper focuses on investigations of the effects on spermatogenesis in male rats following administration over two generations. 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 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 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 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.
Dose / conc.:
0 ppm (nominal)
Remarks:
Concentration in drinking water
Dose / conc.:
25 ppm (nominal)
Remarks:
Concentration in drinking water
Dose / conc.:
100 ppm (nominal)
Remarks:
Concentration in drinking water
Dose / conc.:
175 ppm (nominal)
Remarks:
Concentration in drinking water
Dose / conc.:
250 ppm (nominal)
Remarks:
Concentration in drinking 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:
NOAEC
Effect level:
250 ppm (nominal)
Based on:
test mat.
Remarks:
NaF
Sex:
male
Basis for effect level:
other: No effects were seen on spermatogenesis and endocrine function
Remarks on result:
other:
Remarks:
Concentration in drinking water
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 250 ppm groups were significantly lower than controls. This was considered a random occurrence by the authors because no dose-related effects were observed.
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
250 ppm (nominal)
Based on:
test mat.
Remarks:
NaF
Sex:
male
Basis for effect level:
other: No effects were seen on spermatogenesis and endocrine function
Remarks on result:
other: Concentration in drinking water
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 175 ppm 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 250 ppm group (P generation) was significantly lower than the control group. Spleen weights in the 175 and 250 ppm 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.

In the absence of information on acutal received doses, it is not possible to determine a NOAEL expressed as mg/kg bw/d.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Reliable study used for read across from sodium fluoride to calcium fluoride. The study was conducted according to guidelines considered equivalent to OECD 416
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No data are available for the substance, calcium difluoride, however a number of studies (including high quality NTPA and FDA data) are available for NaF. The much greater water solubility of sodium fluoride (41300 mg/L) compared to calcium fluoride (15 mg/L) means that the bioavailability of fluoride from NaF is likely to be much greater than that of fluoride from CaF2 and therefore represents a worst case.

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. Chinoyet 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. Narayana et al (1994) administered sodium fluoride to male rats by gavage for 50 days and reported adverse effects on sperm count, motility, fertility and sperm morphology. A 70 day recovery period resulted in partial recovery. Zhou et al (2013) examined the effects of sodium fluoride in female rats following administration of sodium fluoride over 6 months via the drinking water. He noticed decreased ovary and uterus weights and deviations in the hormone levels.

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 Messer et al (1973) was due to the influence of fluoride on the 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 data are available for the substance, however a number of studies (including high quality FDA data) are available for NaF. Reliable data do not indicate any specific reproductive or developmental toxicity for inorganic fluoride compounds.
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.

Justification for selection of Effect on fertility via oral route:
No adverse effects were observed at the highest administered dose level for sodium fluoride, which represented a worse case for fluoride exposure than exposure to calcium fluoride, based on differential solubility. Numerous supporting studies are presented to reflect the absence of reproductive effects and to demonstrate the lack of significant effects on specific fertility/reproductive endpoints

Effects on developmental toxicity

Description of key information

A number of studies (including high quality FDA data) are available for NaF. The much greater water solubility of sodium fluoride (41300 mg/L) compared to calcium fluoride (15 mg/L) means that the bioavailability of fluoride from NaF is likely to be much greater than that of fluoride from CaF2 and therefore represents a worst case. 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: NTP study: abstract available
Qualifier:
according to
Guideline:
other: NTP protocol
Qualifier:
equivalent or similar to
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 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 sacrifice
Dose / conc.:
0 ppm (nominal)
Remarks:
Concentration in drinking water
Dose / conc.:
50 ppm (nominal)
Remarks:
Concentration in drinking water
Dose / conc.:
150 ppm (nominal)
Remarks:
Concentration in drinking water
Dose / conc.:
300 ppm (nominal)
Remarks:
Concentration in drinking water
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 wight gain was significantly reduced at 300 ppm 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 300 ppm 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:
18 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
NaF
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: Maternal toxicity
Remarks:
Corresponds to 150 ppm NaF in drinking water
Dose descriptor:
NOAEL
Effect level:
8.14 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
fluoride
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: Maternal toxicity
Dose descriptor:
NOAEL
Effect level:
27 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
NaF
Basis for effect level:
other: No developmental effects observed at highest dose level
Remarks on result:
other: Corresponds to 300 ppm NaF in drinking water
Dose descriptor:
NOAEL
Effect level:
12.21 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
fluoride
Basis for effect level:
other: No developmental effects observed ad highest dose level
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 foetal body weight per litter, or external, visceral, or skeletal malformations.
Remarks on result:
not determinable due to absence of adverse toxic effects
Developmental effects observed:
no

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 300 ppm NaF. Post-exposure water consumption was normal in these animals indicating the probability of decreased palatability of the 300 ppm 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
27 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The developmental toxicity database consists of high quality NTP investigations with other supporting FDA information. The quality of the data for read across from
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

The developmental toxicity of calcium fluoride and sodium fluoride was investigated in the mouse by Fleming & Greenfield (1954). Pregnant female mice were administered the substances in the drinking water or by intraperitoneal injection during gestation. Treatment caused structural changes in the jaw and teeth of offspring, with retardation of skeletal calcification. Calcium fluoride was reported to be more toxic, possible due to the higher fluoride content; high doses of both substances caused resorption or foetal death.

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).

Stratmann et al (1979a,b; 1981) studied the effects of calcium fluoride administration to mice by single intraperitoneal injection on Day 9 of gestation, by dietary administration throughout gestation or over several generations. Embryotoxic effects of calcium fluoride were noted in this study only at very high single intraperitoneal dose levels (1600 mg/kg bw or higher), very high dietary concentrations in the repeated dose phase (20.48g%) or in the generation study (10.24g% and higher). The authors note that the negative effects of the substance on embryogenesis are slight compared to other fluoride compounds and conclude that the substance shows no effects at dose levels relevant for caries prophylaxis and is 'absolutely safe'.


Justification for selection of Effect on developmental toxicity: via oral route:
No key study was identified but read-across information was available from a series of investigations with sodium fluoride. The NTP data set was considered to provide the fullest overall assessment of developmental effects following administration in drinking water

Toxicity to reproduction: other studies

Additional information

Kim et al (2015) incubated mouse spermatozoa with sodium fluoride in media that support in vitro fertilization. The test resulted in observations of reduced intracellular ATP generation, motility and motion kinematics, as well as reduced fertilization and blastocyst formation. The biological relevance of these findings is limited, in particular in light of the available high-quality data indicating no adverse effects in vivo.

 

Two studiers are available that take a more mechanistic point of view. In the study by Zhang et al (2016), male mice were administered sodium fluoride via the drinking water with or without concurrent exposure to SO2 via inhalation. The results showed a decrease in sperm quality and altered morphology and ultrastructure of the blood-testis barrier (BTB), as well as reduced expression levels of some BTB-associated proteins.

In the study by Cao (2016), male mice were treated with sodium fluoride in the drinking water. Observed effects included reduced testosterone content and sperm number and increased sperm malformations. Down-regulation Ssty2 and Sly mRNA and protein was found to occur. The information available in the publication did not allow the determination of actual received fluoride doses.

It is to be noted 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 above.

Justification for classification or non-classification

Reliable data do not indicate any specific reproductive or developmental toxicity for inorganic fluoride compounds. No classification for reproductive toxicity is therefore proposed for calcium difluoride under the CLP Regulation (1272/2008/EC).