Hydrogen fluoride

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Testing the potential of sodium fluoride to affect spermatogenesis in the rat

Author:

Sprando RL, Collins TFX, Black TN, Rorie J, Ames MJ & O'Donnell M

Year:

1997

Bibliographic source:

Food and Chemical Toxicology 35: 881-890

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

The study 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

Test material

Test material information

Constituent 1

Reference substance name:

7681-94-4

IUPAC Name:

7681-94-4

Constituent 2

Reference substance name:

Sodium fluoride

EC Number:

231-667-8

EC Name:

Sodium fluoride

Cas Number:

7681-49-4

Molecular formula:

NaF

IUPAC Name:

sodium fluoride

Details on test material:

Sodium fluoride (CAS 7681-94-4), obtained from Sigma Chemical Co., MO, USA, Lot no. 109F0102. No trace element impurities were detected in the sample. Sodium fluoride (NaF) was the test substance, and the authors were specifically looking at the effects of fluoride. The test substance readily dissociates in aqueous conditions producing fluoride ions. A read across is therefore proposed for hydrogen fluoride as the effects of the fluoride ion in the body will be the same.

Test animals

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).

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

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

Doses / concentrations

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

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 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

250 ppm (nominal)

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)

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.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 ppm (nominal)

Sex:

male

Basis for effect level:

other: Spermatogenesis and endocrine function

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 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.

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.

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.