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Toxicological information

Toxicity to reproduction

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: FDA GLP study published in peer reviewed journal

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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.

Test animals

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%.

Administration / exposure

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).
Doses / concentrations
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

Examinations

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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.