Registration Dossier

Diss Factsheets

Administrative data

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
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study done, published and reviewed by FDA and hence considered as abolutely reliable. Study is done under GLP conditions. Read across justification is given in section 13 assessment reports: WF6 justification for read across

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1998
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
This study provides quantitative information on the effect of sodium fuoride (NaF) on the testes of F1 generation male rats exposed in utero and during lactation to NaF at one of four concentrations (25, 100, 175, 250 ppm). At weaning, the F1 generation males were exposed to NaF in their drinking water for 14 weeks, after which time testicular tissues were perfusion-fixed with glutaraldehyde and observed after being embedded in plastic. The seminiferous tubules comprised 89%, 87%, 88%, 88% and 88% of the total testis volume while the interstitial space occupied 9.3%, 11.2%, 10.2%, 9.8% and 9.9% of the total testis volume for the 0, 25, 100,175 and 250 ppm NaF treatment groups, respectively.
The study design is comparable to a standard two-generation reprodcutive toxicity study, however these papers are focusing on investigations of the effects on spermatogenesis in male rats following administration over two generations.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Sodium fluoride
EC Number:
231-667-8
EC Name:
Sodium fluoride
Cas Number:
7681-49-4
IUPAC Name:
sodium fluoride
Details on test material:
NaF (CAS No. 7681-94-4, Sigma Chemical Co.,
St Louis, MO, USA, Lot No. 109F0102) and a
standard reference material purchased from U.S.
Pharmacopeia (Rockville, MD, USA) were analysed
at the Food and Drug Administration for Na and
F concentrations and trace element contaminants.
Both materials were analysed by instrumental neu-
tron activation analysis (INAA) and prompt-
gamma neutron activation analysis (PGAA), two
techniques that use dierent nuclear reactions and
transitions. The Na and F concentrations of NaF
from the Sigma Chemical Co. and U.S. Pharmacopeia were determined to be the same and
no trace element impurities were detected in either
material by these techniques.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
The 25 male rats utilized in this study were obtained from a larger two-generation reproduction study (see Sprando et al., 1997). The rats were treated as follows: male and female CD VAF + Sprague-Dawley rats (22 days old) obtained from Charles River Laboratories (Raleigh, NC, USA). After a quarantine period of 1 week, the male and female rats were assigned to either a control group (<0.2 ppm NaF) or one of four treatment groups (25, 100, 175 or 250 ppm NaF) by a stratified random experimental assignment procedure. Rats received sodium fluoride in their drinking water ad lib. for approximately 14 weeks: 10 week before mating, during 3 weeks of mating and for approximately 1 week after mating. Males and females within a treatment group were mated. Pregnant P generation females (pregnancy was determined by the presence of sperm plugs in the cage and the presence of sperm in the vagina) continued to be exposed to fluoride (in their drinking water) from day 0 of gestation until the end of lactation. On post-partum day 4, litters were culled to 10 pups (five males and five females) per litter where possible by using a random number table. At day 21, males and females were randomly selected to represent the F1 generation from as many litters as possible. The weanlings remained in the same treatment groups as their parents and were exposed to sodium fluoride for approximately 14 weeks (10 weeks pretreatment, 3 weeks mating, 1 week post-treatment) at which time testicular tissues were collected. During the course of this study rats were housed under standard controlled temperature (67-74°F), humidity (40-70%) and light (12 hr light:12 hr dark) and fed a low fluoride NIH-07 diet (7.95 ppm fluoride) to minimize interference with fluoride in the drinking water. The diet was prepared by Ziegler Bros., Inc. (Gardners, PA, USA) and is the same formulation as that used in the NTP chronic study (NTP, 1990).

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
NaF was dissolved in drinking water, which was passed through a purification system in order to obtain a NaF concentration of 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:
The water used to prepare the fluoride solution for the treated animals and that given to the control rats was obtained by filtering house-distilled water through a Hydro PICOSYSTEMS water purification system. The concentration of fluoride in the PICOSYSTEMS-treated water was determined to be less than 0.2 ppm. NaF 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. NaF concentrations for both the control and treated groups were determined by using a 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 group.
Duration of treatment / exposure:
14 weeks per generation
Frequency of treatment:
daily
Details on study schedule:
Rats received sodium fluoride in their drinking water ad lib. for approximately 14 weeks: 10 week before mating, during 3 weeks of mating and for approximately 1 week after mating. Males and females within a treatment group were mated. Pregnant P generation females (pregnancy was determined by the presence of sperm plugs in the cage and the presence of sperm in the vagina) continued to be exposed to fluoride (in their drinking water) from day 0 of gestation until the end of lactation. On post-partum day 4, litters were culled to 10 pups (five males and five females) per litter where possible by using a random number table. At day 21, males and females were randomly selected to represent the F1 generation from as many litters as possible. The weanlings remained in the same treatment groups as their parents and were exposed to sodium fluoride for approximately 14 weeks (10 weeks pretreatment, 3 weeks mating, 1 week post-treatment) at which time testicular tissues were collected.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
25 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
100 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
175 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
250 ppm
Basis:
nominal in water
No. of animals per sex per dose:
P generation (male):
0 ppm = 12
25 ppm = 13
100ppm = 13
175 ppm = 12
250 ppm = 14

F1 generation (male):
0 ppm = 12
25 ppm = 12
100ppm = 12
175 ppm = 12
250 ppm = 12

The same numbers of female rats were used, but no examinations were performed on these rats.
Control animals:
yes
Details on study design:
After a quarantine period of 1 week, the male and female rats were assigned to either a control group (<0.2 ppm NaF) or one of four treatment groups (25, 100, 175 or 250 ppm NaF) by a stratified random experimental assignment procedure.
On post-partum day 4, litters were culled to 10 pups (five males and five females) per litter where possible by using a random number table. At day 21, males and females were randomly selected to represent the F1 generation from as many litters as possible.
Positive control:
none

Examinations

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 data
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):
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. Examinations in the F1 males were identical to those in the P males.
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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A statistically signifcant difference in body weight was seen between the controls and the NaF-treated rats in the 100ppm, 175ppm and a borderline statistically significant effect was noted in the 250ppm NaF treatment groups. Decrease is not dose related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
A statistically signifcant difference in body weight was seen between the controls and the NaF-treated rats in the 100ppm, 175ppm and a borderline statistically significant effect was noted in the 250ppm NaF treatment groups. Decrease is not dose related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Description (incidence and severity):
Test substance intake: reduction in water consumption in higher dose groups

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
250 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Spermatogenesis and endocrine function

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

liver weights in the 100 and 250 ppm groups were significantly lower than in the control group. The authors considered this as a random occoruence, since there was no relation to the dose.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Spermatogenesis

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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 25 ppm group. The right testis weight of the F1 generation males was significantly higher than that of the P generation males in the 100 ppm 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 175 ppm group were compared. Prostate/seminal vesicle weights were not significantly different between treated and control rats in either generation.

A statistically signifcant difference in body weight was seen between the controls and the NaF-treated rats in the 100 ppm, 175 ppm and a borderline statistically significant effect was noted in the 250 ppm NaF treatment groups. Decrease is not dose related. A reduction in water consumption in higher dose groups was observed. Indicating that dosing was at the limit.

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.

Applicant's summary and conclusion

Conclusions:
Prolonged exposure to sodium fluoride in drinking water did not adversely affect spermatogenesis or endocrine function in two generations of male rats.
Hypothetical NOAEL for WF6: 520 mg/m³
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 250 ppm. 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 250 ppm groups, and in the testicular capsule in the 100 ppm 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.
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

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 250 ppm. 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 250 ppm groups, and in the testicular capsule in the 100 ppm 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.

250 ppm of HF equals 42 ppm of WF6, which is corresponding to 520 mg/m³ at 20°C and 1,013 bar.