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Toxicological information

Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
After dissolution and disociation in environmental (aqueous)/physiolological media the constituents of tin difluoride become bioavailable and the overall toxicity of the dissociated substance can be described by the toxicity of the individual constituents/ions. Since synergistic effects are not to be expected, the human health and environmental hazard assessment of the assessment entity tin difluoride consists of an individual assessment of the assessment entities tin cation and the fluoride anion. The tin cation and the fluoride anion are considered to represent the overall toxicity of tin difluoride in a manner proportionate to the fluoride and the metal (represented by one of its readily soluble salts). Based on the above information, unrestricted read-across is considered feasible and justified.
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Developmental toxicity of sodium fluoride was assessed in rats that were given drinking water containing five different dose levels throughout gestation (days 0 to 20). Reproductive parameters included pregnancy rate, implantation efficiency, and foetal viability. Foetal development parameters included foetal body weight and growth measurements, number of runts and number of runts/litter. Developmental parameters included external variations, sternebral variations, soft tissue variations, and externsive skeletal variations.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CD-CRL:CD-BR, VAR+
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Wilmington, MA, USA
- Weight at study initiation: 351-375 g (males) and 175-200 g (females)
- Housing: stainless steel cages in stainless steel racks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7-25.5
- Humidity (%): 15-73
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Animals were exposed to sodium fluoride in drinking water at concentrations of 0, 10, 25, 100, 175 or 250 ppm.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From gestation day 0 to gestation day 20
Frequency of treatment:
ad libitum in drinking water
Duration of test:
terminated on gestation day 20
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
175 ppm (nominal)
Dose / conc.:
250 ppm (nominal)
No. of animals per sex per dose:
34 females/0 ppm
35 females/1.4 ppm
33 females/3.9 ppm
33 females/15.6 ppm
33 females/24.7 ppm
35 females/25.1 ppm
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The same three dose levels ( 25, 100 and 175 ppm) evaluated in the NTP carcinogenic study were used with the addition of a higher (250 ppm) and lower (10 ppm) dose level to obtain a broader range of toxic effects.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 6, 9, 12, 15, 18 and 20


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: gestation days 0, 3, 6, 9, 12, 15, 18 and 20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Other: Average percentage early + late deaths/litter
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
Clinical signs in control and treated rats were analyzed by Fisher's exact test. Feed consumption and fluid consumption of control and treated rats were analyzed by analysis of variance (ANOVA) followed by a protected least significant difference (LSD) test (two-tailed). Since some of the fluid consumption values were unusually large, a Grubb's outlier test was performed. Body weight gain data were analyzed by ANOVA followed by a protected LSD test (two-tailed). The numbers of corpora lutea, implants, viable foetuses, viable males and viable females per litter were subjected to ANOVA followed by a protected LSD test (one-tailed). Data for implantation efficiency and the average percentage of early deaths, late deaths and total resorptions per litter were transformed by the Freeman-Tukey arc-sine transformation and analysed by ANOVA followed by protected LSD test (one-tailed). For the percentage of dams not pregnant, percentage of litters with one or more or two or more resorptions and specific external foetal variations, a Fisher's exact test (one-tailed) was used to compare control and treated groups. Foetal weights and crown-rump lengths were analyzed by a nested ANOVA followed by a protected LSD test (two-tailed). For incidence of foetal sternebral, skeletal or soft-tissue variations, both specific and combined, the number of foetuses with a least one, at least two and at least three variations per litter were transformed by a Freeman-Tukey arc-sine transformation. The transformed data were anyalysed by ANOVA followed by a protected LSD test (one-tailed). A Fisher's exact test 9one-tailed) was used to analyse the proportion of litters with foetuses having at least one, at least two and at least three sternebral, skeletal or soft-tissue variations.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Females drinking water with 175 ppm sodum fluoride drank significantly less fluid and ate less food than did the controls during gestation (the amount of sodium fluoride ingested daily was 24.7 mg/kg/day). Females drinking water with 250 ppm sodium fluoride drank significantly less fluid and ateless food than did controls thoughout gestation (the amount of sodium fluoride ingested daily was 25.1 mg/kg/day). These findings were reflected by random decreases in body weight gain in rats at 175 ppm and significant decreases in body weight gain in rats at 250 ppm.
Dose descriptor:
NOEC
Effect level:
175 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no

Details on embryotoxic / teratogenic effects:
A significant increase in the average number of foetuses with three or more skeletal variations and the number of litters with foetuses with three or more skeletal variations was increased in the 250 ppm group. However, there were no dose-related increases in the incidence of soft tissue variations, external anomalies, or effects on the development of specific bones, including sternebrae.
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
No definative treatment-related effects on foetal growth or on the incidence of external, visceral, or skeletal abnormalities at levels of sodium fluoride up to and including 250 ppm (25.1 mg/kg/day) were found.
Reason / purpose:
reference to other study
Reference
Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
After dissolution and disociation in environmental (aqueous)/physiolological media the constituents of tin difluoride become bioavailable and the overall toxicity of the dissociated substance can be described by the toxicity of the individual constituents/ions. Since synergistic effects are not to be expected, the human health and environmental hazard assessment of the assessment entity tin difluoride consists of an individual assessment of the assessment entities tin cation and the fluoride anion. The tin cation and the fluoride anion are considered to represent the overall toxicity of tin difluoride in a manner proportionate to the fluoride and the metal (represented by one of its readily soluble salts). Based on the above information, unrestricted read-across is considered feasible and justified.
Reason / purpose:
reference to other study
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
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:
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.


Dose / conc.:
0 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
250 ppm (nominal)
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).
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.
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.
Remarks on result:
not determinable due to absence of adverse toxic effects
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
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.

Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
not specified
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.
Reason / purpose:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
After dissolution and disociation in environmental (aqueous)/physiolological media the constituents of tin difluoride become bioavailable and the overall toxicity of the dissociated substance can be described by the toxicity of the individual constituents/ions. Since synergistic effects are not to be expected, the human health and environmental hazard assessment of the assessment entity tin difluoride consists of an individual assessment of the assessment entities tin cation and the fluoride anion. The tin cation and the fluoride anion are considered to represent the overall toxicity of tin difluoride in a manner proportionate to the fluoride and the metal (represented by one of its readily soluble salts). Based on the above information, unrestricted read-across is considered feasible and justified.
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Weight at study initiation: mean body weights/group ranged from 248 to 253 g
- Fasting period before study: no
- Diet (e.g. ad libitum): ad libitium
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 (average)
- Humidity (%): 55% (average)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Animals were exposed to 0, 50, 150 or 300 ppm sodium fluoride in drinking water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preexposure analysis of drinking water solutions demontrated the concentrations of sodium fluoride to be within a range of 104-107% of target concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Any other deviations from standard protocol: The morning on which sperm were found in a vaginal lavage was designated as GD 0.
Duration of treatment / exposure:
Gestation days 6 through 15
Frequency of treatment:
ad libitum - drinking water
Duration of test:
Terminated on Gestation day 20
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
150 ppm (nominal)
Dose / conc.:
300 ppm (nominal)
No. of animals per sex per dose:
26
Control animals:
yes
Details on study design:
- Dose selection rationale: The sodium fluoride concentrations tested were 50, 150 and 300 ppm which corresponded to approximately 7.5, 22.5, and 45 mg/kg/day based on an estimated water consumption of 150 mL/kg body weight/day. The LD50 for sodium fluoride in rats is at least 52 mg/kg/day or approximately 350 ppm if administered in drinking water. The concentrations were chosen after consideration of data from the NTP 14-day study in which female rats given 400 ppm sodium fluoride in drinking water showed decreasesd body weight and water consumption and clinical signs of toxicity (dehydration , lethargy, and hunched posture) while 800 ppm killed all the animals. The high dose (300 ppm) was chosen in an effort to induc e some maternal toxicity while avaoiding the potentially confounding effects of dehydration seen at the 400 ppm level in the 14-day study. The 300 ppm level was anticipated to be a nonlethal exposure based on the lack of lethality among rats exposed to 300 ppm sodium fluoride in the NTP 6-month drinking water studty and 50 ppm low level was expected to produce no maternal toxicity based on a lack of effects related to sodium fluoride consumption in the NTP 2-year toxicity study at exposures up to 100 ppm.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily beginning on gestation day 6


BODY WEIGHT: Yes
- Time schedule for examinations: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: liver, kidney and uterus


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
General linear model (GLM) procedures were applied for the analyses of varience (ANOVA) of maternal and fetal parameters. Prior to GLM-ANOVA analysis, an arcsine-square root transformation was performed on all litter-derived percentage data to normalize the means and Bartlett's test for homogeneity of variance was performed on all data to be analyzed by ANOVA. GLM-ANOVA anyalysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects, and dose x replicate interactions. When ANOVA revealed a significant (p<0.05) dose effect, Dunnett's test and Williams' test were used to compare treated to control groups. One-tailed tests wre used for all pairwise comparisons except maternal body and organ weeights, food and water consuumption , fetal body weight, and percentage of males per litter. If a significant (p<0.05) dose x replicate interaction occurred, then the data for that endpoint were analyzed separately for dose effects witnin each replicate in the study, as well as for all replicates combined. Nominal scale measures were analyzed by a X2 test for independence and by a test for linear trend on proportions. When a X2 test showed significant experinent-wise differences, a one-tailed Fisher's exact test was used for pairwise comparisons of treatment and control groups.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No animals died during the course of the study. No treatment-related clinical signs were observed in confirmed-pregnant animlsa during or after administration of sodium fluoride. Maternal body weight gain during the first 2 days of exposure (GD 6 to 8) was significantly reduced at 300 ppm relative to controls. Maternal water consumption of rats given 300 ppm sodium fluoride was significantly decreased for each period of observation from GD 6 to 15. Maternal food consumption was also decreased in the animals given 300 ppm sodium fluoride from GD 8 to 10, but did not differ from controls for any other period of measurement. Maternal liver and kidney weights on GD 20 were not differenct from control . Examination of uteri demonstrated that 100% (26/26), 96% (25/26), 89% (23/26), and 96% (25/26) of the mated animals in the control throught high-dose groups were pregnant.
Dose descriptor:
NOAEC
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No embryotoxic or teratogenic effects related to exposure to sodium fluoride in drinking water was observed at any dose concentration.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Maternal exposure to sodium fluoride at conentrations up to 300 ppm (approximately 27 mg/kg/day) during organogenesis (gestation days 6 to 15) did not significantly affect the frequency of postimplantation loss, mean fetal body weight/litter, or external, visceral or skeletal malformations in the rat.

Data source

Reference
Reference Type:
publication
Title:
Developmental toxicity of sodium fluoride measured during multiple generations
Author:
Collins, TFX, R.L. Sprando, T.N. Black, M.E. Shackelford, N. Olejnik, M.J. Ames, J.I. Rorie and D.I. Ruggles
Year:
2001
Bibliographic source:
Food and Chemical Toxicology 39: 867-876.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The developmental effects of continous exposure to 0, 25, 100, 175 or 250 ppm sodium fluoride in drinking water over three generations of rats were evaluated.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): (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)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Willmington, MA, USA
- Weight at time of receipt: 51-75 g for males and females
- Weight at study initiation: (P) Males: 100.5 - 100.9 g; Females: 93.9 -94.8 g
- Housing during exposure period: Single animals were housed in stainless-steel cages suspended in stainless-steel racks. Pregnant females 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.
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.
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
Duration of treatment / exposure:
P and F1 generation male and female rats were dosed for 10 weeks prior to mating, and females were dosed throughout gestation.
Frequency of treatment:
continuous, 7 days per week
Duration of test:
Effects of sodium fluoride in drinking water on developmental toxicity was measured during the parental (P) generation and two filial (F1 and F2) genereations.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
175 ppm (nominal)
Dose / conc.:
250 ppm (nominal)
No. of animals per sex per dose:
The P generation contained 48 male and 48 female rats per group that were dosed for a 10 week period before mating. Eight mated P females per group were selected for caesarean sections on gestation day 20 to assess development effects of sodium fluoride on F1 generation litters. The remaining P generation female rats were allowed to litter and wean their F1 pups. On postnatal day 21, 36 F1 male and 36 F1 female pups were randomly selected to continue being dosed for a 10 week period before being allowed to mate and provide F2 generation litters. At gestation day 20, the mated F1 females were observed for the last time, euthanized, and caesarean sections were performed on the F2 generation litters to assess development effects of sodium fluoride.
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

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: Initial body weight on gestation day 0 and at termination on gestation day 20


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Initial measurement on gestation day 0 and at termination on gestation day 20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: no data


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
F1 Fetuses
- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

F2 Fetuses
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
For feed consumption, number of corpora lutea, implants, total alive, male and female alive, an analysis of variance (ANOVA) was performed. If the ANOVA indicated a significant effect, then a least significant difference (LSD) test was run to compare the control with each treated group. For implantation efficiency and early, late and total (early + late) resorptions, the data were first transformed using a Freeman-Tukey arcsine transformation followed by an ANOVA. If the ANOVA indicated a significant effect, then a LSD test was performed. For body weight gain and gravid uterine weight, an analysis of covariance (ANCOVA) was performed. If the ANCOVA showed a significant effect, a LSD test was run to compare the control with each treated group. For fetal weight and crown-rump measurements, a nested ANOVA followed by a LSD test was performed on the groups if the nested ANOVA showed a significant effect. For the incidences of clinical signs in male and female rats, and specific soft-tissue, sternebral, and skeletal variations in fetuses, a Fisher's exact test was used to compare treated groups to the control group. The same test was used to compare the control with each treated group for the number of litters with at least one, at least two, and at least three soft-tissue, sternebral and skeletal variations. For the average number of fetuses per litter with at least one, at least two, and at least three soft-tissue, sternebral or skeletal variations, the data were first transformed by a Freeman-Tukey arcsine transformation. The transformed data were then analyzed using an ANOVA followed by a LSD test. For the average number of ossified vertebrae in fetuses, a nested ANOVA was performed on the data. If the nested ANOVA results were significant, a LSD test was performed to compare the control with each treated group.
Historical control data:
Developmental effects of sodium fluoride on rats from an earlier study by Collins et al., 1995 are referred to in the current study.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects: no effects

Effect levels (maternal animals)

Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Clinical Findings & Morphological Development: Clinical findings were unremarkable during gestation of P and F1 females. Morphological development of F1 and F2 fetuses was similar in all groups.
Maternal and Reproductive Findings: Reproduction of either P or F1 dams was not affected by the sodium fluoride doses. In P and F1 dams, the mean number of corpora lutea, mean number of implants, implantation efficiency, mean number of viable fetuses, and average percentage of early and late deaths per litter showed no dose-related differences. The number of P and F1 females with one and with two early plus late deaths (combined) was similar in all groups. Sex distribution of F1 and F2 fetuses showed no dose-related response. No F1 litters were totally resorbed. Three F2 litters were totally resorbed, one litter each in the 0, 25 and 175 ppm groups. Fetal body weight and crown-rump length of F1 and F2 fetuses were not affected. A single statistically significant decrease in crown-rump length of F2 females at 175 ppm was considered random. Very few runts were observed among the F1 and F2 litters, and the incidence of runts was not dose-related.
Fetal Skeletal Development Analysis: The number of F2 fetuses with incompletely ossified, non-ossified, bipartite, or malformed sternebrae was similar in control and treated groups. The number of litters containing fetuses with malaligned sternebrae was greater in all treated groups than in the control group, but the increases were not dose-related. One fetus with fused sternebrae in the control group was considered random. When the incidence of sternebral variations in fetuses and the number of fetuses with at least one, at least two, and at least three variations per litter were analyzed, no dose-related increases were seen. The significant increase in the percentage of litters with at least one variation at 175 ppm was considered random due to lack of dose relationship. As an additional measure of ossification deficiency, data on the individual sternebrae that were bipartite or showed reduced or lack of ossification were combined and analyzed; no dose-related effects were seen. The following single occurrences were considered random: one fetus with 15 ribs (0 ppm), one fetus per group with reduced ossification of the ischium (0, 25, 175 ppm), one fetus with reduced ossification of the scapula (100 ppm), and one fetus per group with reduced ossification of the humerus (175 ppm). Sodium fluoride did not affect the ossification of caudal vertebrae. Decreased ossifcation of the hyoid bone was observed at 175 and 250 ppm, and the ossifiction at 250 ppm was statistically significant. When the incidence of skeletal variations was analyzed by fetus and by litter, no dose-rrelated significant differences were observed. Vertebral ossification, also considered a measure of fetal ossification, showed no dose-related effects.
Fetal Visceral Development Analysis: No dose-related changes were noted in the development of fetal soft tissues. The significant increases in the incidence of moderate hydroureter at 25 ppm and 100 ppm, and the incidence of moderate enlargement of the ureter at the kidney at 25 ppm were considered random because of the lack of dose relationship. When the incidence of soft-tissue variations was analyzed by fetus and by litter, no dose-related changes were noted in any treated group. The following single occurrences were considered random: one fetus with moderate edema (25 ppm), one fetus with ectopic kidney (25 ppm), one fetus with enlarged kidney (100 ppm), and one fetus with four vessels off the aortic arch (100 ppm).

Effect levels (fetuses)

Dose descriptor:
NOEC
Effect level:
175 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A concentration of 175 ppm sodium fluoride in drinking water of a parental (P) and two filial generations (F1 and F2) of rats did not produce developmental effects and is considered a no-observed effect concentration for developmental toxicity. A concentration of 250 ppm sodium fluoride in drinking water of a parental (P) and two filial generations (F1 and F2) of rats caused statistically significant decreased ossification of the hyoid bone in F2 fetuses and is considered an effect concentration for developmental toxicity.