Registration Dossier

Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test parameters evaluated in this study do not totally comply with a specific test guideline, but are well documented and scientifically acceptable. The study was conducted accordiing to Good Laboratory Practice regulations.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2001

Materials and methods

Principles of method if other than guideline:
The effects of fluoride ingestion in drinking water were measured during three generations of rats.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): sodium fluoride (NaF); CAS No. 7681-49-4
- Physical state: solid
- Analytical purity: as pure as the standard reference sample purchased from the U.S. Pharmacopeia, Rockvile, MD
- Impurities (identity and concentrations): no trace element impurities were detected
- Lot/batch No.: 109F0102

Test animals

Species:
rat
Strain:
other: caesarean -derived (CD CRL:CD-BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Willmington, MA, USA
- Weight at study initiation: (P) Males: 100.5 - 100.9 g; Females: 93.9 -94.8 g
- Housing: Single animals were housed in stainless-steel cages suspended in stainless-steel racks. Pregnant females and females with litters (to weaning) were housed in polycarbonate tubs with Sani-Chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 - 22.8
- Humidity (%): 35-67
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
- Animals were exposed to sodium fluoride in drinking water at concentrations of 0, 25, 100, 175 or 250 ppm.
- Drinking water used in the study was obtained by filtering house-distilled water through a Hydro Pico pure water system.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: no longer than 1 week; females that failed to mate after 1 week were remated to a different male within the same group
- Proof of pregnancy: presence of sperm in vaginal lavage referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: yes - Each female was allowed up to 3 weeks for mating.
- After successful mating each pregnant female was caged in a polycarbonate tub with Sani-Chips
- Any other deviations from standard protocol: At gestation day 20, caesarean sections were performed on 8 P females per group. The dams and their offspring were examined, and the results are reported by Collins, T.F.X. et al. (2001). After mating, P adult males were transferred to a separate protocol for a study of the effects of sodium fluoride on male reproduction.
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 determined at the FDA 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. Sodium fluoride concentrations were determined each time dosing solutions were prepared for any treatment group, including the control.
Duration of treatment / exposure:
P and F1 male and female rats were treated for 10 weeks before mating.
Frequency of treatment:
continuous; 7 days/week
Details on study schedule:
- F1 parental animals were not mated until 10 weeks after being selected from the P litters.
- Selection of parents from F1 generation occured when pups were 21 days of age.
- Age at mating of the mated animals in the study: Approximately 13 weeks old
- At gestation day 20, caesarean sections were performed on pregnant adult P and F1 females. The dams and their offspring were examined and the results are reported by Collins, T.F.X. et al. (2001).
- At weaning on postnatal day 21, 10 F1 males and 10 F1 females from each group were selected and given fluoride solution to postnatal day 90. Growth and feed and fluid consumption of these F1 animals were measured during postnatal days 21-90. These animals were referred to as the F1 non-mated subset.


Doses / concentrations
Remarks:
Doses / Concentrations:
Sodium fluoride concentrations of 0, 25, 100 or 250 ppm in drinking water.
Basis:
nominal in water
No. of animals per sex per dose:
48 for the P generation and 36 for the F1 generation
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The 25, 100 and 175 ppm sodium fluoride doses were based on results of the National Toxicology Program (NTP 1990) chronic 2-year toxicology and carcinogenicity study in rats and mice. The 250 ppm sodium fluoride dose was based on results of a developmental study in rats conducted in the current study's laboratory in which rats tolerated this dose without adverse effects (Collins et al. 1995).

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: At sudy initiation and 10 weeks for P animals. At gestation day 21 and 10 weeks for F1 animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: At sudy initiation and 10 weeks for P animals. At gestation day 21 and 10 weeks for F1 animals.



Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 13-14 pups (males/females combined)/litter; excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number of pups (male/female combined), number born alive, number alive on day 4 (pre-culling and post-culling), and number of alive on postnatal days 7, 14 and 21, presence of gross anomalies, and weight gain.


GROSS EXAMINATION OF DEAD PUPS: No information
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: 10 P males and 10 F1 males from each group were sacrificed soon after the last litters in each generation were produced.
- Maternal animals: 10 P and 10 F1 females from each group were sacrificed after the last litter of each generation was weaned.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. All gross lesions were recorded. Necropsies of remaining animals (P post-lactation females), P post-mating males, F1 extra males, and F1 non-pregnant females) were performed by the Developmental and Subchronic Toxicology Team at FDA and are not presented in this report.


HISTOPATHOLOGY / ORGAN WEIGHTS
- The animals were weighed and the following tissues were weighed: thymus, heart, kidneys, adrenal glands, brain, liver, testes, epididymides, prostate, seminal vesicle, ovaries and spleen.
-Histopathological examination of the following tissues was performed: heart, aorta, spleen, thymus, lungs, liver, kidney, pituitary and adrenal glands, thyroid gland, parathyroid gland, trachea, esophagus, stomach, duodenum, pancreas, jejunum, ileum, cecum, colon, testes, ovaries, urinary bladder, epididymides, seminal vesicle (base), prostate, uterus (bifurcation), cervix, vagina, eyes with optic nerves, mammary gland (right thoracic), sternum with marrow, brain (cerebellum, pons/medull, cerebrum), spinal cord (cervical, thoracic, lumbar), and any gross lesions. In addition, the following tissues were evaluated for animals from the control and high-dose groups (250 ppm): salivary gland, tongue, mesenteric lymph node, rectum, intraorbital lacrimal glands, psoas muscle, skin, skiull (2 sections), Harderian glands, teeth (upper incisors and upper molars), nasal turbinates, vertebral column (caudal-thoracic and caudal-lumbar), right femur with marrow and right sciatic nerve.
Postmortem examinations (offspring):
SACRIFICE
- F1 offspring (10 males and 10 females) from each group that were not selected as parental animals were sacrificed at 21 days of age.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. All gross lesions were recorded.


HISTOPATHOLOGY / ORGAN WEIGHTS
- The F1 weanling animals were weighed and the following tissues were weighed: thymus, heart, kidneys, adrenal glands, brain, liver, testes, epididymides, prostate, seminal vesicle, ovaries and spleen.
-Histopathological examination of the following tissues was performed: heart, aorta, spleen, thymus, lungs, liver, kidney, pituitary and adrenal glands, thyroid gland, parathyroid gland, trachea, esophagus, stomach, duodenum, pancreas, jejunum, ileum, cecum, colon, testes, ovaries, urinary bladder, epididymides, seminal vesicle (base), prostate, uterus (bifurcation), cervix, vagina, eyes with optic nerves, mammary gland (right thoracic), sternum with marrow, brain (cerebellum, pons/medull, cerebrum), spinal cord (cervical, thoracic, lumbar), and any gross lesions. In addition, the following tissues were evaluated for animals from the control and high-dose groups (250 ppm): salivary gland, tongue, mesenteric lymph node, rectum, intraorbital lacrimal glands, psoas muscle, skin, skiull (2 sections), Harderian glands, teeth (upper incisors and upper molars), nasal turbinates, vertebral column (caudal-thoracic and caudal-lumbar), right femur with marrow and right sciatic nerve.
Statistics:
Clinical signs in control and treated rats were analyzed by Fisher's Exact Test. For parameters of feed consumption (two-tail), number of implants, total animals alive, males alive and females alive (one-tail), an analysis of variance (ANOVA) was performed. If the ANOVA indicated a significant effect (P equal to or less than 0.05), then a least significant difference (LSD) test was run to compare the control with each treated group. For the parameters body weight, organ weight, body weight gain, and gravid weight an analysis of covariance was performed. If the analysis of covariance showed a significant effect, a LSD test (two-tail) was run to compare the control with each treated group. Analyses of organ to body weight and organ to brain weight ratios were performed using body weight and brain weight as the covariate, respectively. For statistical analysis of mean fluid consumption, each dose group was first checked for outliers using the Grubbs outlier test (one-tail) because several animals (distributed throughout all groups) played with their water tubes, causing the fluid to drip which led to unusually and misleadingly high fluid consumption values for these animals. If an animals was deemed an outlier from the Grubbs test, it was removed from the statistical analysis of fluid consumption. Each time period for each dose group was then tested for outlying values and if a value was determined to be an outlier, a replacement value was calculated using a method described by Snedeco and Cochran (1967).
Reproductive indices:
For females of both the P and F1 generation, the following parameters were recorded: the number exposed to mating, the number of sperm-positive, the number producing a litter, mating index, fertility index #1 (number producing a litter/number of sperm-positive) X 100, fertility index #2 (number producing a litter/number exposed to mating) X 100, and the time to mating (days plus or minus S.E.M.).

For males of both F0 and F1 generations, the following parameters were recorded: the number exposed to mating, the number producing plugs or sperm-positive females, the number producing a litter, mating index, fertility index #1 (number producing a litter/number producing sperm-positive females) X 100, and fertility index #2 (number producing a litter/number exposed to mating) X 100.
Offspring viability indices:
The parameters used to evaluate survival of total F1 offspring (male and female) included: number of implants, number born, number born alive, number alive on day 4 (pre-culling and post culling), and number alive on days 7, 14 and 21.

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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- No dose-related clinical effects were observed in either adult males or females from the P and F1 generations. During the 10-week growth period, one P male died from the 25 ppm group, and one F1 male died from the control group. No other animals died during the growth period.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- Mean body weights of females and males in the P and F1 generations were similar in all groups. No statistically significant effects were seen in any group. Weight gain of P females and males during days 0-70 showed a significant negative linear regression. Only the individual weight gain of P males in the 250 ppm group was statisically significantly less than the control. In the F1 generation, weight gain of females and males was similar; no significant dose-related effects were observed.
-No dose-related changes in body weight gain were observed in P females during gestation and lactaction. Gravid body weight of F1 females also showed no relation to dose.
- During the 10-week growth period, there were no significant differences in feed consumption in P females between the control and treated groups. During the same period, there was a significant decrease in overall food consumption by P males in the 250 ppm group. This was due to significantly less feed consumption by the 250 ppm rats than by the control group during the first 7 weeks and week 9 (8 of the 10 weeks during the growth period). Overall mean feed consumption of F1 females showed a significant negative linear regression for days 0-70, although none of the values was significantly less than the control value. During the same period, F1 males in the treated groups consumed less feed than did the control group, but the decreases were neither dose-related nor significant.
- Feed consumption was measured during gestation and lactation of P females that gave birth and reared their litters, and during gestation of F1 females. During gestation, no dose-related changes were observed in feed consumption in either P or F1 females. During lactation, feed consumption of P females was similar in control and all test groups.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
- During the 10-week growth period, the P and F1 females and males in the 175 and 250 ppm groups drank significantly less than the animals in their respective control groups. F1 males in the 100 ppm group also drank significantly less than the control group. Decreased consumption was attributed to decreased palatability.
- Sodium fluoride consumption by P females ranged from 3.5 to 27.3 mg NaF/kg/day at 25 to 250 ppm, respectively, and consumption in F1 females was similar (3.8 to 28.0 mg NaF/kg/day, respectively, for 25 to 250 ppm). Sodium fluoride consumption by P males ranged from 2.8 to 23.1 mg NaF/kg/day at 25 to 250 ppm, respectively, and consumption in F1 meles ranged from 3.0 to 24.1 mg NaF/kg/day, respectivley, for 25 to 250 ppm.
- With respect to fluoride consumption, P females consumed 1.5 to 12.3 mg F/kg/day, and F1 females consumed similar amounts (1.7 to 12.7 mg F/kg/day, respectively, for 25 to 250 ppm). P males consumed 1.3 to 10.5 mg F/kg/day, and F1 males consumed similar amounts (1.4 to 10.9 mg F/kg/day, respectively, for 25 to 250 ppm.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
- The reproductive P generation female mating indices were over 90% in all groups. P female fertility indices were decreased slightly (but not significantly) at 250 ppm. Average time to mating was less in treated groups than in the controls, but the differences were not dose-related.
- Mating indices of F1 females were also over 90% indicating lack of sodium fluoride-related effects. The fertility indices of the females in the 25 and 250 ppm groups were slightly less than the control animals, but the differences were not statistically significant and are probably due to random variation. No dose-related effect was seen in the average time to mating of F1 females. The longest time to mating was seen in the 25 ppm group.
- Mating indices of P males were not dose-related. The mating indices for P males were slightly lower than those in P females, although all values were over 90%. The lowest male fertility index occurred in the 250 ppm group, but the difference was not statistically significant. Two males in the 25 ppm group mated with two females each, but neither produced a litter; no other group contained males that mated without consistantly producing a litter. The lack of dose relationship and the lack of statistical significance indicated that these decreases are probably random.
- All the mating indices of F1 males weere less than those of the P males; there were no dose-related decreases. Fertility indices of F1 males were lower in the 25 and 250 ppm groups than in any other group. Because of the lack of dose response, these effects appeared to be random.


ORGAN WEIGHTS (PARENTAL ANIMALS):
In P females, no significant differences in organ weights were observed between control and treated groups. In P adult females weighing 320 +/- 11.1 g, organ weights were: thymus 0.38 +/- 0.04 g; heart 1.26 +/- 0.03 g; liver 15.18 +/- 0.71 g; spleen 0.63 +/- 0.03 g; both kidneys 2.78 +/-0.08 g; both adrenals 0.08 +/- 0.01 g; both ovaries 0.17 +/- 0.01 g; and brain 1.96 +/- 0.03 g. Body weight of P control adult males was 546.6 +/- 19.4 g and organ weights were: thymus 0.78 +/-0.11 g; heart 1.64 +/- 0.07 g; liver 22.75 +/- 1.24 g; spleen 0.98 +/- 0.05 g; both kidneys 4.20 +/- 0.12 g; both adrenals 0.05 +/- 0.00 g; both testes 3.49 +/- 0.06 g; brain 2.16 +/- 0.02 g; both epididymides 1.44 +/- 0.09 g; seminal vesicle 1.53 +/- 0.20 g; and protate 1.24 +/- 0.13 g. Organ-to-body-weight rations and organ-to-brain weight ratios were calculated for all adult female and male organs, and no dose-related or significant effects were seen.


GROSS PATHOLOGY (PARENTAL ANIMALS):
In the F1 generation, no stained or mottled teeth were observed in either sex of any group. However, whitening of the teeth was observed in both females and males in the 100, 175 and 250 ppm sodium fluoride groups. Although the effect was mild, a number of affected females and males was increased in a dose-related, statiistically significant manner. Animals in the 25 ppm group were not affected.

HISTOPATHOLOGY (PARENTAL ANIMALS):


OTHER FINDINGS (PARENTAL ANIMALS):
- In 10 F1 weanlings of each sex per group that were allowed to grow to day 90 (the non-mated subset), no clinical change attributable to sodium fluoride was observed in either females or males. Overall mean feed consumption of females and males showed no significant dose-related change. Mean body weight and body weight gain of treated females and males also showed no significant dose-related changes.
- Overall mean fluid consumption by F1 females and males was decreased at 175 and 250 ppm in a dose-related manner, but only the consumption at 250 ppm was significantly reduced. The amounts of sodium fluoride and fluoride consumed by the females and males were similar to those consumed by the P and F1 animals during their 10-week growth period. The amount of sodium fluoride consumed at 250 ppm was 28.4 mg/kg/day for females and 27.1 mg/kg/day for males. Of this, fluoride consumption was 12.8 mg/kg/day for females and 12.3 mg/kg/day for males.

Effect levels (P0)

Dose descriptor:
NOEC
Remarks:
reproduction
Effect level:
ca. 250 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: A lack of dose response and the lack of any statistically significant effects in mating indices indicate that prolonged exposure to sodium fluoride in the drinking water did not adversely affect female or male reproduction in the P generation.

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 specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Histopathological findings:
effects observed, treatment-related

Details on results (F1)

VIABILITY (OFFSPRING)
Survival indices of F1 offspring (implantation, live-birth, day-4 survival, day-7 survival, day-14 survival and lactation indices) were calculated for combined female and male offspring, as well as female and male offspring separately. Neither signiificant nor dose-related effects were observed.

CLINICAL SIGNS (OFFSPRING)
Growth and development of F1 female and male pups to postnatal day 21 were similar in all groups.

BODY WEIGHT (OFFSPRING)
In F1 male and female F1 pups, no significant dose-related mean body weight changes were found. However, on day 0, a significant mean body weight increase occurred in F1 female pups dosed with 100 ppm sodium fluoride when compared to controls.


ORGAN WEIGHTS (OFFSPRING)
In F1 weanling males and females, no significant dose-related effects were observed in the organ-to-body-weight ratios or in the organ-to-brain-weight rations. In F1 weanling control females weighing 74.8 +/- 2.0 g, organ weights were: thymus 0.35 +/- 0.01 g; heart 0.41 +/- 0/01 g; liver 3.57 +/- 0.08 g; spleen 0.30 +/- 0.02 g; both kidneys 0.95 +/- 0.04 g; both adrenals 0.02 +/- 0.00 g; both ovaries 0.05 +/- 0.01 g; and brain 1.46 +/- 0.04 g. In F1 weanling control males weighing 82.0 +/- 2.5 g, organ weights were: thymus 0.36 +/- 0.02 g; heart 0.45 +/- 0.01 g; liver 3.99 +/- 0.15 g; spleen 0.35 +/- 0.02 g; both kidneys 1.04 +/- 0.04 g; both adrenals 0.03 +/- 0.00 g; both testes 0.51 +/- 0.03 g; brain 1.54 +/- 0.07 g; both epididymides 0.13 +/- 0.03; seminal vesicle 0.04 +/- 0.01; and prostate 0.09 +/- 0.01 g.


HISTOPATHOLOGY (OFFSPRING)
Histomorphological tissue alterations attribuable to administration of sodium fluoride consisted of an increase in the development of prominant growth lines (basophilic lines in the dentin and enamel of the tooth) in upper incisors of 8 female and 10 male F1 weanlings that received 250 ppm compared to the controls. In weanling animals, the growth lines were graded minimal in 7 males and 5 females and mild in 6 rats, 3 of each sex. No other histological findings were noted.

Effect levels (F1)

Dose descriptor:
NOEC
Remarks:
reproduction
Generation:
F1
Effect level:
ca. 250 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: A lack of dose response and the lack of any statistically significant effects in mating indices indicate that prolonged exposure to sodium fluoride in the drinking water did not adversely affect female or male reproduction in the F1 generation.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Sodium fluoride in drinking water of Sprague-Dawley rats at levels up to 250 ppm (equivalent to 28.4 mg sodium fluoride/kg body weight/day or 12.8 mg fluoride/kg body weight/day) had no adverse effects on reproduction throughout three generations. No cumulative toxic effects were observed in the three generations.