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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 (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: FDA GLP study published in peer reviewed journal
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: CD CRL:CD-BR
Sex:
male/female
Details on test animals 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.
Estrous 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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: estrous 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Species:
rat
Quality of whole database:
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.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
No data are available.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
No data are available.
Additional information

Published studies

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.

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 sodium hydrogen difluoride 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.

Justification for selection of Effect on fertility via oral route:
This study is of standard design, is well reported and includes an assessment of dietary fluoride exposure.

Effects on developmental toxicity

Description of key information
No studies with sodium hydrogen difluoride 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
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: NTP study: abstract available
Reference:
Composition 0
Composition 0
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
Test material information:
Composition 1
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 Day6-15 of gestation and sacrific
Remarks:
Doses / Concentrations:
0, 50, 150, or 300 ppm
Basis:
nominal in 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 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.
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
Species:
rat
Quality of whole database:
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.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
No data are available.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
No data are 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 selection of Effect on developmental toxicity: via oral route:
High quality study performed in the preferred species for this endpoint.

Justification for classification or non-classification

The substance will form HF under physiological conditions, with subsequent dissociation to the constituent (hydrogen, sodium and fluoride) ions. The toxicologically relevant component of the substance is considered to be fluoride, and systemic toxicity following repeated oral and inhalation exposure may result from fluoride. Reliable studies do not indicate any developmental toxicity or reproductive toxicity of fluoride.

No classification is therefore proposed for reproductive or developmental toxicity according to CLP criteria .