Magnesium fluoride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Justification for type of information:

Read-across justification is provided in Appendix 3 of the CSR.

The available toxicological data on the target and source substances is outlined in the data matrix (Table 8).
The toxicological properties of the target substance are related to the magnesium cations (Mg2+) and fluoride anions (F-). Thus, data on other magnesium salts (mainly magnesium chloride) and inorganic fluorides (mainly sodium fluoride) are justified in the human health hazard assessment.
Humans take in between 250 and 350 mg/day of magnesium and need at least 200 mg, but the body deals very effectively with this element, taking it form food when it can, and recycling what we already have when it cannot. There is no evidence that magnesium produces systemic poisoning although persistent over-indulgence in taking magnesium supplements and medicines can lead to muscle weakness, lethargy and confusion.
Chloride is relatively not toxic to human health but does pose organoleptic issues. Accordingly, EPA has set the potable limit for chloride in drinking water at 250 mg/L.

Experimental data obtained with the magnesium chloride indicate that it has low oral toxicity after repeated exposure. The available data does not show any test item related adverse effects on the highest doses (> 2000 mg/kg bw/day) tested. Sodium fluoride administration did not cause test item related adverse effects in rats dosed up to 175ppm for 2 years. Only exceptions were abnormalities in the teeth of rats in the two highest dose groups (100 and 175 ppm sodium fluoride).

Sodium is a compound of many foodstuffs, for instance of common salt. It is necessary for humans to maintain the balance of the physical fluids system. Sodium is also required for nerve and muscle functioning. Thus, there is no evidence that sodium produces systemic poisoning at reasonably possible exposures.

Fluoride has both positive and negative effects on human health, but there is a narrow range between intakes that are associated with these effects. Exposure to all sources of fluoride, including drinking-water and foodstuffs, is important There is little information to characterize the dose–response relationships for the different adverse effects. In particular, there are few data on total exposure, particularly with respect to intake and fluoride absorption. The most serious effect is the skeletal accumulation of fluoride from long-term excessive exposure to fluoride and its effect on non-neoplastic bone disease — specifically, skeletal fluorosis and bone fractures. There is clear evidence from India and China that skeletal fluorosis and an increased risk of bone fractures occur at total intakes of 14 mg fluoride/day and evidence suggestive of an increased risk of bone effects at total intakes above about 6 mg fluoride/day.
The data of repeated dose toxicity and reproduction toxicity are proposed to be used for read-across according to the information requirements of Annex VIII.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Justification for type of information:

Read-across justification is provided in Appendix 3 of the CSR.

The available toxicological data on the target and source substances is outlined in the data matrix (Table 8).
The toxicological properties of the target substance are related to the magnesium cations (Mg2+) and fluoride anions (F-). Thus, data on other magnesium salts (mainly magnesium chloride) and inorganic fluorides (mainly sodium fluoride) are justified in the human health hazard assessment.
Humans take in between 250 and 350 mg/day of magnesium and need at least 200 mg, but the body deals very effectively with this element, taking it form food when it can, and recycling what we already have when it cannot. There is no evidence that magnesium produces systemic poisoning although persistent over-indulgence in taking magnesium supplements and medicines can lead to muscle weakness, lethargy and confusion.
Chloride is relatively not toxic to human health but does pose organoleptic issues. Accordingly, EPA has set the potable limit for chloride in drinking water at 250 mg/L.

Experimental data obtained with the magnesium chloride indicate that it has low oral toxicity after repeated exposure. The available data does not show any test item related adverse effects on the highest doses (> 2000 mg/kg bw/day) tested. Sodium fluoride administration did not cause test item related adverse effects in rats dosed up to 175ppm for 2 years. Only exceptions were abnormalities in the teeth of rats in the two highest dose groups (100 and 175 ppm sodium fluoride).

Sodium is a compound of many foodstuffs, for instance of common salt. It is necessary for humans to maintain the balance of the physical fluids system. Sodium is also required for nerve and muscle functioning. Thus, there is no evidence that sodium produces systemic poisoning at reasonably possible exposures.

Fluoride has both positive and negative effects on human health, but there is a narrow range between intakes that are associated with these effects. Exposure to all sources of fluoride, including drinking-water and foodstuffs, is important There is little information to characterize the dose–response relationships for the different adverse effects. In particular, there are few data on total exposure, particularly with respect to intake and fluoride absorption. The most serious effect is the skeletal accumulation of fluoride from long-term excessive exposure to fluoride and its effect on non-neoplastic bone disease — specifically, skeletal fluorosis and bone fractures. There is clear evidence from India and China that skeletal fluorosis and an increased risk of bone fractures occur at total intakes of 14 mg fluoride/day and evidence suggestive of an increased risk of bone effects at total intakes above about 6 mg fluoride/day.
The data of repeated dose toxicity and reproduction toxicity are proposed to be used for read-across according to the information requirements of Annex VIII.

Reason / purpose for cross-reference:

reference to other study

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 extensive skeletal variations.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test was conducted on a read-accross substance sodium fluoride (CAS 7681-49-4; EC 231-667-8).

Species:

rat

Strain:

not specified

Remarks:

CD-CRL:CD-BR, VAR+

Details on test animals or test system and environmental conditions:

Animals were obtained from Charles River Laboratories, Inc., Wilmington, MA, USA.
Weight at study initiation: 351-375 g (males, not treated with sodium fluoride) and 175-200 g (females).
Housing: stainless steel cages in stainless steel racks.
Diet: Females were fed with low fluoride NIH-07 diet (7.95 ppm fluoride) available ad libitum
Water: Weight/volume sodium fluoride solutions were prepared in Aqua Cool Ultra Pure water (Ionics, Inc., Watertown, MA, USA), available 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:

Females were mated 2:1 with males. Cohabitation started 16.30 at each mating day and lasted overnight. In the morning females were removed from the cages and tested fro successful mating by smearing for the precense of sperm in the vaginal lavage. Females that had mated were presumed pregnant.

Duration of treatment / exposure:

From gestation day 0 to 20

Frequency of treatment:

ad libitum in drinking water

Duration of test:

All animals were terminated on gestation day 20

Remarks:

0, 10, 25, 100, 175 or 250 ppm sodium fluoride (equivalent to 0, 1.4, 3.9, 15.6, 24.7 or 25.1 mg/kg bw, respectively)

No. of animals per sex per dose:

35 females/0 ppm, 35 females/10 ppm, 35 females/25 ppm, 36 females/100 ppm, 37 females/ 175 ppm, 37 females/250 ppm.

Control animals:

yes, concurrent vehicle

Details on study design:

Female rats were administered with 0, 10, 25, 100, 175 or 250 ppm sodium fluoride solution in drinking water ad libitum during gestation days 0 to 20. 35-37 animals were used per dose group. On GD 20 females were killed and caesarean sections were performed. Uteri were opened and examined in situ.

Maternal examinations:

The animals were observed daily throughout the study for clinical signs and behavioural changes. Body weight was measured on gestation days 0, 3, 6, 9, 12, 15, 18 and 20. Water consumption was measured every 3 days (gestation days 0, 3, 6, 9, 12, 15, 18 and 20) and feed consumption was measured on gestation days 7, 14 and 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination, icluding presence and position of resorption sites, implantation sites and live or dead featuses. Number of corpora lutea were counted and recorded.

Fetal examinations:

All featuses were weighed, sexed, measured for crown, rump length and examined for the presence of external abnormalities. Alternately featuses were examined for skeletal abnormalities or for soft-tissue abnormalities.

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS: No clinical signs of toxicity were recorded during the study.
MORTALITY: No animals died during the study.
BODY WEIGHT: Significant decreases in body weight gain were seen in the females of the 250-ppm group on days 0-3 and 6-9 and overall on days 0-20.
FOOD CONSUMPTION: Females dosed with 250 ppm sodium fluoride ate significantly less feed throughout gestation than the control animals.
WATER CONSUMPTION AND COMPUND INTAKE: Animals in the 175 ppm dose group drank significantly less fluid than in the control group. Also the females in the 250 ppm dose group consumed significantly less fluid than the ones in the control group throughout gestation. Owing to the significantly lower fluid consumption in the animals at the 175 and 250 ppm dose levels, a relatively small difference was seen in the average amount of NaF ingested daily by the animals at these two dose levels. The amounts of sodium fluoride ingested daily were 1.4, 3.9, 15.6, 24.7 and 25.1 mg/kg bw.
BEHAVIOUR: No changes in behaviour were recorded.

Dose descriptor:

NOAEL

Effect level:

ca. 175 ppm

Based on:

test mat.

Basis for effect level:

other: Developmental toxicity

Dose descriptor:

NOAEL

Effect level:

ca. 175 ppm

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity

Details on embryotoxic / teratogenic effects:

Foetal growth was not affected by sodium fluoride. 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.

Dose descriptor:

NOAEL

Effect level:

175 ppm

Sex:

male/female

Basis for effect level:

skeletal malformations

Developmental effects observed:

no

Conclusions:

No definitive 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.

Data source

Reference

Reference Type:

publication

Title:

Developmental toxicity evaluation of sodium fluoride administered to rats and rabbits in drinking water

Author:

Heindel, Jerrold J., Hudson K. Bates, Catherine J. Price, Mellissa C. Marr, Christina B. Meyers and Bernard A. Schwetz

Year:

1996

Bibliographic source:

Fundamental and Applied Toxicology 30, 162-177

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium fluoride

EC Number:

231-667-8

EC Name:

Sodium fluoride

Cas Number:

7681-49-4

Molecular formula:

FNa

IUPAC Name:

sodium fluoride

Specific details on test material used for the study:

Test was conducted on a read-across material sodium fluoride (CAS 7681-49-4; EC 231-667-8).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Cesarean-originated, barrier sustained Crl:CD (BR) VAF/Plus outbred albino rats were obtained from Charles River Laboratories, Inc., Raleigh, NC.
Sperm-positive females were individually housed in solid-bottom polycarbonate cages with stainless-steel wire lids and Ab-Sorb-Dri cage litter.
NIH-07 Rodent chow and either deionized/fileterd water or the prepared sodium fluoride-spiked drinking water were available ad libitum throughout gestation.
12:12 hr light:dark cycle; average temperature 22 degrees celcius and average humidity 55%. All environmental conditions were monitored and controlled by computer.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Sodium fluoride was administered to rats in the drinking water, provided ad libitum.

Details on analytical verification of doses or concentrations:

The method detection limit was used to determine the level of NaF present in the control water, and this value was used to calculate the drinking water doses. The amount of F present in the standard diet was also determined.

Details on mating procedure:

After a 7-day quarantien period, individual breeding pairs of rats were cohabited overnight. In the first replicate study there was three consecutive breeding dates and five consecutive breeding dates in the second. The morning on which sperm were found in vaginal lavage was designated as Gestational Day (GD) 0.

Duration of treatment / exposure:

Timed-mated rats were allowed ad libitum access to deionized/filtered drinking water containing 0, 50, 150 or 300 ppm sodium fluoride from GD 6 to 15 (i.e. treated water was removed on the morning of GD 16).

Frequency of treatment:

Drinking water was available ad libitum.

Duration of test:

All animals were terminated on GD 20. On the second replicate of the study, 10 female rats were added and they were killed on GD 16 for blood collection.

Doses / concentrations

Remarks:

0, 50, 150 or 300 ppm

No. of animals per sex per dose:

26 female rats per dose were used for the developmental toxicity study and an additional 10 rats per dose were used for blood collection. The study was performed in two replicates.

Control animals:

yes, concurrent vehicle

Details on study design:

26 pregnant female rats per dose were used for the developmental toxicity study and an additional 10 rats per dose were used for blood collection. Two replicates of the study were performed. Body weight, feed and water consumption development were followed during the study. Animals were terminated on GD 20. On the second replicate of the study, 10 female rats were added and they were killed on GD 16 for blood collection in order to determine serum fluoride levels. Gross pathology was performed for the maternal bodies. Gross pathology and histopahtological examinations were performed on featuses.

Examinations

Maternal examinations:

Animals were observed daily for clinical signs of toxicity. Food and water intakes and body weights were recorded on gestation days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. All animals were sacrificed on gestation day 20 and examined for maternal body and organ weights (liver, right kidney and intact uterus), implant status, foetal weight, sex and morphological development. An additional 10 mated animals per groups were subjected to the same experimental regimen but sacrificed on gestation day 16 for blood collection for determination of serum fluoride concentration.

Ovaries and uterine content:

Uterine contents were examined - implant status, foetal weight, sex and morphological development were recorded.

Fetal examinations:

Foetuses were examined for external, visceral or skeletal malformations, in addition to foetal body weights and sex.

Statistics:

General linear models (GLM) procedures were applied for the analyses of variance (NOVA) of maternal and fetal parameters. GLM-ANOVA analysis determined the significance of dose-response relationship 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 were used for all pairwise comparisons except maternal body and organ weights, food and water consumption, fetal body weight and percentage of males per litter.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS: No treatment related clinical signs were observed in confirmed-pregnant animals during or after administration of sodium fluoride.
MORTALITY: No animals died during the course of the study
BODY WEIGHT: Maternal body weight gain during the first 2 days of exposure (GD 6 to 8) was significantly reduced at 300 ppm relative to controls. The mean maternal body weight gain for the treatment period as a whole exhibited a decreasing trend, and the comparison among groups for an effect of dose approached statistical significance.
WATER CONSUMPTION: Maternal water consumption of the animals dosed with 300 ppm sodium fluoride was significantly decreased for each period of observation from GD 6 to 16. 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.
ORGAN WEIGHT: Maternal liver and kidney weights on GD 20 were not different from control.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

300 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: this is the highest dose tested, no clear test item related adverse effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Sodium fluoride exposure did not affect the number of corpora lutea, implantations, live featuses or the percentage of early deaths or late fetal deaths per litter. In addition, no significant effects on external, visceral, or skeletal malformations were found.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

300 ppm

Sex:

male/female

Basis for effect level:

other: No addverse effects were observed. This is the maximum dose tested

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Control water fluoride levels were <0.6 ppm NaF.

Food contained an average of 12.4 ppm F (11.6 -13.4 ppm F).

The calculated doses from drinking water were 7, 18 and 27 mg NaF/kg bw/d (3, 8 and 12 mg F/kg bw/d) for the low, intermediate and high-dose groups respectively. Intake from food added approximately 2 mg NaF/kg bw/d (1 mg F/kg bw/d) to the intake for each group.

Determination of serum fluoride levels in the 10 animals per group terminated on 16 revealed mean levels of 0.007 ± 0.002, 0.035 ± 0.040, 0.039 ± 0.039, and 0.187 ± 0.076F at the end of the exposure period.

Applicant's summary and conclusion

Conclusions:

In conclusion, sodium fluoride in drinking water at doses up to 300 ppm rats throughout major organogenesis caused no definitive developmental toxicity.