Barium fluoride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Barium fluoride will dissociate under physiological conditions to form barium and fluoride ions. Since no data on the reproduction toxicity are available for barium fluoride, the available studies with sodium fluoride and barium chloride were used to assess the toxicity of Ba2+ and F- ions. The calculated oral NOAEL of barium fluoride based on the NOAEL of fluoride (49 mg/kg bw/day) is lower than the calculated NOAEL of barium fluoride based on the NOAEL of barium (229 mg/kg bw/day) therefore it is concluded that the toxicological moiety for the reproduction toxicity of BaF2 is fluoride.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: FDA GLP study published in peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD CRL:CD-BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

CD-BR VAF rats, obtained from Charles River Laboratories, USA. Males ad females weighed 51-75g on receipt. They were acclimatised for 1 week, and identified individually by ear tags. Rats were fed low-fluoride NIH-07 diet (7.95ppm fluoride, Ziegler Bros., USA). Single animals were housed in stainless steel cages suspended in racks. Pregnant females and females with litters were housed in polycarbonate tubs with Sani-Chips as bedding. Light in the animal room was provided on a 12 h light/dark cycle. The average temperature was 71-73oF, and average humidity was 35-67%.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

NaF was administered in drinking water. Weight/volume NaF solutions were prepared in water obtained by filtering house-distilled water through a Hydro Pico pure water system. The concentration of fluoride in the filtered water was <0.2ppm.

Details on mating procedure:

Rats in the F0 generation were mated on a 1:1 basis. Females that failed to mate after 1 week were remated to a different male within the same treatment group. Each female was allowed up to 3 weeks for mating. Cohabitation began at approximately 16.30h on each mating day. Mating was confirmed by the presence of sperm in the vaginal lavage. Females that mated were presumed pregnant. Rats were randomly selected from the resultant F1 generation for mating following the same procedure as in the F0 generation. Litter mates were not mated. The day mating was confirmed was designated as gestation day 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

NaF concentrations for the control and treated groups were determined by potentiometric titration of the fluoride ion with a fluoride ion electrode by using an EA 940 pH/ISE meter with appropriate electrodes and filling solutions for fluoride analysis. NaF concentrations were determined each time dosing solutions were prepared for any treatment group including the control.

Duration of treatment / exposure:

10 weeks.

Frequency of treatment:

Daily.

Details on study schedule:

F0 parental animals were exposed to NaF for 10 weeks. They were then mated randomly within treatment groups. At gestation day 20 8 females per group were subject to caesarean section and examination, these results were reported elsewhere. The remaining females were allowed to litter and wean their pups to postnatal day 21 (the day of birth was designated postnatal day 0). On postnatal day 4 litters were culled to 10 pups by random procedure.

On postnatal day 21 36 F1 males and 36 F1 females per group were randomly selected for mating (no more than 2 of each sex per litter). After 10 weeks NaF exposure they were allowed up to 3 weeks to mate. At gestation day 20, caesarean sections were performed on the pregnant females (the results are discussed elsewhere).

Remarks:

Doses / Concentrations:
0, 25, 100, 175, or 250 ppm
Basis:
nominal in water

No. of animals per sex per dose:

F0 generation: 48 rats per sex per dose
F1 generation: 36 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were based on the National Toxicology Program (1990) chronic two year study, plus an additional higher dose (250ppm) based on a developmental toxicity study conducted previously by the authors (Collins et al 1995).

Positive control:

Not examined

Parental animals: Observations and examinations:

Body weights were recorded during the 10 week exposure period.
All animals were examined individually on a dialy basis for clinical signs and mortality.
Feed and water consumption were measured during the 10 week exposure period.

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Not examined as part of this study (reported elsewhere).

Litter observations:

The number of still born pups was noted. On postnatal days 0, 4, 7, 14 and 21 each pup was observed, sexed and weighed. Litters were culled to 10 pups per litter on postnatal day 4. The incidence of runts was calculated on postnatal days 0, 4, 7, 14 and 21 (the weight of individual pups was compared to the average of litter averages per sex, any animal weighing less tha 70% of the grand mean weight was termed a runt).

Postmortem examinations (parental animals):

Ten males and females from each group (F0 adults, F1 weanlings and F0 adults) were evaluated for histopathological effects. All gross lesions were recorded, animals were weighed, and organ weights were determined for the: thymus, heart, kidneys, adrenal glands, brain, liver, testes, epididymides, prostate, seminal vesicle, ovaries and spleen. Histopathology was performed on the following tissues for all animals: heart, aorta, spleen, thymus, lungs, liver, kidney, pituitary, adrenal glands, thyroid and parathyroid, trachea, oesophagus, stomach, duodenum, pancreasm jejunum, ileum, cecum, colon, testes, ovaries, urinary bladder, epididymides, semival vesicle, prostate, uterus, cervix, vagina, eyes with optic nerves, mammary gland, sternum with marrow, brain, spinal cord, and all gross lesions. In addition, the following tissues were evaluated for animals in the control and 250ppm groups: salivary gland, tongue, mesenteric lymph node, rectum, intraorbital lacrimal glands, psoas muscle, skin, skull, Harderian glands, teeth, nasal turbinates, vertebral column, right femur with marrow and right sciatic nerve. All tissues were observed from sections stained in haematoxylin and eosin.

Postmortem examinations (offspring):

See above.

Statistics:

Clinical signs were analysed by Fishers Exact test. Feed consumption, and reproductive data were analysed using ANOVA. LSD tests were used to compare controls with each treated group. Body weight and weight gain, organ weights and ravid weight were compared between groups using ANCOVA and LSD tests.
Fluid consumption was contraindicated by several animals that played with the water tubes. Outliers were removed from the statistical analysis using the Grubb's test.
P values of less than or equal to 0.05 were considered significant.

Reproductive indices:

Mating index - (no. sperm positive/no. exposed to mating) x100
Fertility index #1 - (no. produced litter/no. sperm positive) x100
Fertility index #2 - (no. produced litter/no. exposed to mating) x100

Offspring viability indices:

Number of live births, runts, growth.

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

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

No dose-related clinical effects were observed in either males or females.

There was a significant decrease in overall feed consumption by F0 males in the 250ppm group. Rats in the 175 and 250ppm groups drank significantly less than controls. This decreased consumption is attributed to decreased palatability. Weight gain of males and females showed a significant negative linear regression, however only the individual weight gain of the 250ppm males was statistically significantly less than controls.

Female mating indices were over 90% in all groups. Female fertility indices were decreased slightly in the 250 ppm group, but not significantly. Average time to mating was less in treated groups than in the controls but the differences were not dose related.

Mean water consumption per pregnant female was decreased in the 250ppm group during the entire period of gestation.
There were no effects on organ weights or organ to body weight ratios.
There was an increase in the development of prominent growth lines in the upper incisors of all rats that received 250ppm. Hyperkeratosis of hte limiting ridge of the stomach was diagnosed in a small number of rats from the 100, 175 and 250ppm groups.

Dose descriptor:

NOAEL

Effect level:

250 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on reproduction were seen at the highest dose level

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

No dose-related clinical effects were observed in either males or females.

Oveall mean feed consumption of F1 females showed a significant negative linear regression for days 0-70 although none of the values were significantly less than controls. F1 males in the treated groups ate less than the control group but the decreases were neither dose related nor significant. Rats in the 175 and 250ppm groups drank significantly less than controls. F1 males in the 100ppm group drank significantly less than the control group. This decreased consumption is attributed to decreased palatability.
Mating indices of females were over 90%. The fertility indices of the females in thhe 25 and 250ppm were slightly less than controls, but not significantly and probably due to random variation.
All the mating indices of the F1 males were less than those of the F0 males, but there was no dose-related decrease.
Mean fluid consumption per pregnant female was decreased in the 250ppm group during the entire period of gestation. Fluid consumption was significantly decreased in 17 5 ppm group females during the entire period of gestation.
Survival of F1 offspring to postnatal day 21 showed no dose-related effects. Growth and development were similar in all groups, and female and male runts were randomly distributed among the control and treatment groups.
There were no effects on organ weights or organ to body weight ratios.
There was an increase in the development of prominent growth lines in the upper incisors of all rats that received 250ppm. Hyperkeratosis of hte limiting ridge of the stomach was diagnosed in a small number of rats from the 250ppm group.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on reproduction were seen at the highest dose level

Reproductive effects observed:

no

NaF consumption by F0 females ranged from 3.5-27.3 mg NaF/kg bw/d at 25 -250 ppm respectively, and consumption in F1 females was 3.8 -28.0 mg NaF/kg bw/d. Consumption in the F0 males was 2.8 to 23.1 mg NaF/kg/d, and in the F1 males was 3.0 -24.1 mg NaF/kg bw/d.

Conclusions:

Sodium fluoride administered in the drinking water for 10 weeks at dose levels up to 250ppm had no adverse effects on reproduction in rats.

Executive summary:

The effects of sodium fluoride ingestion at 0, 25, 100, 175 or 250 ppm in drinking water was measured in a two-generation study. Reproduction was not affected by NaF administration, and offspring viability also remained unaffected. The decreased fluid consumption noted in high dose groups was attributed to decreased palatability. Mating, fertility and survival indices were not affected. Sodium fluoride caused an increase in the incidence of whitening of tooth enamel, in a dose-related manner from males and females in the 100, 175 and 250ppm groups. There was an increase in the development of prominant growth lines in the upper incisors of F0 and F1 adult rats and F1 weanlings. The reproductive NOAEL in rats was therefore 250 ppm.

Reference 2

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study well documented, meets generally accepted scientific principles, acceptable for assessment. Data and Rating according to the SIDS 2005 on barium cabonate. Deviances when comparing to OECD421: dosing only prior to mating, no individual animal data/tables provided, histopathologic examination, data on food consumption only provided for core study animals, no humidity, sex of pups, and data on stability of test substance in vehicle given. Only the average results of the controls and the high dose groups of each species were available.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In parallel with a subchronic toxicity core study, a premating study was performed with separate groups of rats and mice. Premating exposure period with Barium chloride dihydrate was 60 days for males and 30 days for females.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: Fischer 334/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonson Laboratories, Gilroy, CA
- Age at study initiation: (P) 32 days
- Housing: five per cage in drawer-type polycarbonate cages (shelves covered with filter sheets, bedding, cages and water bottles were changed twice a week, feeders once a week, racks and filters every other week); after 60 days of exposure, the males were placed in individual cages and one female receiving the same dose level (but exposed for 30 days) was cohabited with the male. After mating the females were separated.
- Diet: NIH-o7 pellets (Ziegler Brothers, Gardners, PA)
- Water: ad libitum (dosed or undosed) for 92 consecutive days
- Acclimation period: 10 to 11 days (quarantined)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24 °C
- Air changes (per hr): 13.5 room vol.
- Photoperiod (hrs dark / hrs light): 12/12 (fluorescent lighting)

- no further significant details stated

Route of administration:

oral: drinking water

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Solutions were made weekly in 19-liter quantities by dissolving weighed portions of the test substance in glass-distilled water.
- Concentration in vehicle: 0, 1000 and 4000 ppm
- no further significant details stated

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female rat
- Length of cohabitation: up to one week
- Proof of pregnancy:Examination of microscopic evidence of sperm in vaginal swab every morning
- When evidence of mating was found, the females was separated from the male.
- After mating determinations were made on the eighth day of cohabition and all remaining pairs were separated.
- No remating was performed although pregnancy rate was low.
- no further significant details stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed on all levels before and after use, and at the beginning and midway through the test period. The concentrations were within 1 to 6 % of the theoretical concentration. Method of analysis was not stated.

Duration of treatment / exposure:

Premating exposure period: 60 days for males and 30 days for females

Frequency of treatment:

continuous

Details on study schedule:

- no further significant details stated

Remarks:

Doses / Concentrations:
1000 ppm BaCl2 x 2H2O
Basis:
nominal in water
calculated average dose: 63.5 mg Ba/kg bw/d to males and 64.5 mg Ba/kg bw/d to females

Remarks:

Doses / Concentrations:
2000 ppm BaCl2 x 2H2O
Basis:
nominal in water
calculated average dose: 112 mg Ba/kg bw/d to males and 114 mg Ba/kg bw/d to females

Remarks:

Doses / Concentrations:
4000 ppm BaCl2 x 2H2O
Basis:
nominal in water
calculated average dose: 201.5 mg Ba/kg bw/d to males and 179.5 mg Ba/kg bw/d to females

No. of animals per sex per dose:

20 male and 20 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Other: No remating was performed due to restriction in the study dosing schedule/design.
- no further significant details stated

Positive control:

not required

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly and females were weighed when evidence of mating was found an on the day of parturition.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice weekly


OTHER:
- determination of pregnancy rates in dosed and control animals
- determination of average gestation period
- no further significant details stated

Oestrous cyclicity (parental animals):

- Evaluation of vaginal cytology was performed among treated and control groups.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
- An evaluation of sperm morphology, density, and motility and sperm count was performed among treated and control groups.
- no further significant details stated

Litter observations:

PARAMETERS EXAMINED
The following examinations were performed in F1 offspring:
- pups were examined at birth and day 5
- number of live litter, average litter size at day 0 and 5, pup survival to day 5, pup weight at birth and day 5, external abnormalities

GROSS EXAMINATION OF DEAD PUPS:
- yes, dead pups were examined for external abnormalities

- no further significant details stated

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: not stated
- Maternal animals: All surviving animals were terminated on days 96 and 97.

GROSS NECROPSY
- The vagina, cervix, oviducts, and ovaries were grossly examined and the implantation sites in the uteri were counted.
- The male reproductive organ weights (testis, epidimymal) was determined among treated and control groups.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Complete histologic exams were performed in the parallel animal groups which were not used for reproductive and fertility assessment.

- no further significant details stated

Postmortem examinations (offspring):

- no data

Statistics:

Each parameter for which individual values were available was subjected to a linear least squares regression over the doselevels and the direction of the slope and the p value indicating the significance of the deviation of the slope from 0 was determined. Group means and standard deviations or standard errors were calculated for continuous variables. The multiple comparison procedure of Dunnett (1955) was used for comparison between dosed and control groups. Further, Fisher's extract test, the Cochran-Armitage test (Armitage, 1971; Gart et al., 1979) was used as well as repeated measures analysis of variance (WInter, 1971) and a multivariate analysis of variance (Morrison, 1976).

Reproductive indices:

- no significant details stated

Offspring viability indices:

- no significant details stated

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- one pregnant dam in the 4000 ppm group was terminated in a moribund state 21 days after mating


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- only determined for core study animals


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- There were no treatment-related effects of barium chloride dihydrate on vaginal cytology up to 4000 ppm.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- There were no treatment-related effects of barium chloride dihydrate on epididymal sperm count, sperm motility, sperm morphology, up to 4000 ppm.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- The pregnancy rates were below generally accepted norms: from 40 % in the controls and 65 % in the 4000 ppm group.
- All pregnant dams produced litters except for one in the 4000 ppm group, which was terminated
- The average gestation period of surviving dams was 22 to 22.5 days (in various groups).
- The number of implants per pregnant dam was marginally reduced in the 4000 ppm group compared with the controls (but without statistical significance at p<0.05)


ORGAN WEIGHTS (PARENTAL ANIMALS)
- no effect could be detected on testis or epididymal weight


GROSS PATHOLOGY (PARENTAL ANIMALS)
- necropsy of the terminated dam revealed 7 fetuses and one resorption site

- no further significant details stated

Dose descriptor:

NOAEL

Effect level:

4 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fertility impairment

Dose descriptor:

NOAEL

Effect level:

201.5 mg/kg bw/day (nominal)

Based on:

other: barium

Sex:

male

Basis for effect level:

other: fertility impairment

Dose descriptor:

NOAEL

Effect level:

179.5 mg/kg bw/day (nominal)

Based on:

other: barium

Sex:

female

Basis for effect level:

other: fertility impairment

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- Pup survival to day 5 was 99 % or greater in all treatment groups.

CLINICAL SIGNS (OFFSPRING)
- No external abnormalities were observed in the rat offspring.

BODY WEIGHT (OFFSPRING)
- Rats receiving 4000 ppm exhibited significant although marginal reductions in pup weights at birth (5.20 +/- 0.06 g compared to 5.70 +/- 0.09 g); a comparision of pups weight on day 5 showed no significant differences. Weight gain was comparable among all pup groups.

OTHER
- The average litter size at birth and on postpartum day 5 was marginally reduced in the 4000 ppm group compared with the controls (but without statistical significance at p<0.05)

- no further significant details stated

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

4 000 ppm (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: development toxicity, but the NOAEL is of limited value to evaluate the potential for barium to induce developmental effects

Reproductive effects observed:

not specified

Only the average results of the controls and the high dose groups of each species were available.

No-observed-adverse-effect level (NOAEL) for fertility impairmentwas 4,000ppm in rats. These NOAEL values correspond to average doses of 201.5 and 179.5 mg Ba/kg bw/d to male and and female rats, respectively.

A NOAEL on developmental toxicity of 4,000 ppm is also reported. However, the NOAEL is of limited value to evaluate the potential for barium to induce developmental effects. The reason for this limitation is based on the fact that the premating study design did not include exposure of female animals during the gestational period to barium chloride. Therefore, the premating study has to be considered as an inadequate study of developmental toxicity and cannot be used to determine the occurrence of developmental toxicity.

Conclusions:

Taken together all data of this study, there are no indications of a substantial impairment of fertility in rats up to the highest dose tested. Thus, the NOAEL was 4000 ppm (to average doses of 201.5 and 179.5 mg Ba/kg bw/d to male and and female rats, respectively). No-observed-adverse-effect levels (NOAELs) on developmental toxicity for rats of 4000 ppm were derived from this study. However, this NOAEL is of limited value to evaluate the potential for barium to induce developmental effects because there was no exposure of the females during gestation.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

49 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproduction toxicity study is available on barium fluoride and therefore this endpoint was assessed in a "Weight-of-Evidence" approach in accordance with section 1.2 of REACH Annex XI. Under physiological conditions barium fluoride will dissociate into barium and fluoride ions. Therefore the toxicity of barium fluoride may reasonably be considered to be determined by the availability of Ba2+ cations and F- anions. Comprehensive data on developmental toxicity are available for barium chloride and sodium fluoride. As a first surrogate for bioavailability, the water solubility of a test substance may be used. Both barium chloride and sodium fluoride are highly water soluble with ca. 375 g/L and 40 g/L, respectively at neutral pH, whereas barium fluoride has a solubility of 1.6 g/L. Hence, any read across from barium chloride and sodium fluoride to BaF2 is inherently conservative.

Fluoride

From a two-generation study by Collins et al. (2001) it can be concluded that no fertility effects occurred in rats exposed to NaF in the drinking water at concentrations up to 250 ppm. The dose levels (expressed on a mg/kg bw/d basis) for P and F1 animals were about equal, and based on drinking water consumption data and body weights, the exposure level of 250 ppm is on average equal to 10.7 mg F-/kg bw/d or 12.5 mg F-/kg bw/d for males and non-pregnant females respectively (corresponding to 49 and 58 mg BaF2/kg bw/d, resp.).

Barium

For barium chloride a reproduction screening study is available that was conducted in parallel with a subchronic toxicity study (Dietz et al., 1992). In this study, groups of 20 male and 20 female F344/N rats were exposed to barium chloride dihydrate concentrations of 0, 1000, 2000 or 4000 ppm in drinking water for up to 60 days. Body weight changes and water consumption was measured at regular intervals. After completion of the exposure period, males and females from the same dosage group were housed together until there was evidence of mating or until the end of the mating period (8 days). There were no indications of a substantial impairment of fertility in rats up to the highest dose tested. Thus, the NOAEL was 4000 ppm corresponding to average doses of 201.5 and 179.5 mg Ba/kg bw/d to male and and female rats, respectively (i.e. 257 and 229 mg BaF2/kg bw/d).

Effects on developmental toxicity

Description of key information

Barium fluoride will dissociate under physiological conditions to form barium and fluoride ions. Since no data on the developmental toxicity are available for barium fluoride, the available studies with sodium fluoride and barium chloride were used to assess the effects on developmental toxicity of Ba2+ and F- ions.

From three well-performed embryo- and developmental toxicity studies with NaF an overall NOAEL for maternal toxicity and developmental effects of 11.1 mg F-/kg bw/d was derived corresponding to 51.3 mg BaF2/kg bw/day. Barium dichloride dihydrate was evaluated in a GLP compliant oral prenatal toxicity study according to OECD guideline 414. In absence of developmental effects the NOAEL for developmental toxicity is reported as re-calculated barium fluoride (i.e., NOAEL: ≥ 103 mg BaF2/kg bw/day). Therefore it is concluded that the toxicological moiety for the developmental toxicity of BaF2 is fluoride.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Published study, similar to guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

The animals were Caesarean-derived, viral anitbody-free (CD-CRL:CD-BR, VAF+) rats obtained from Charles River Laboratories, MA, USA. On arrival the males weighed 351-375g, and the females weighed 175-200g. During the study, rats were housed in stainless steel cages suspended in stainless steel racks. Light was provided on a 12 hour light/dark cycle (light 07.30-19.30hr). The temperature was 17.8-25.6oC (64-78oF). Humidity ranged from 15-73%; the lowest humidity value (15%) was recorded once during the first week of the study. During the remainder of the study the range of low humidity values was 24-42%. During mating rats were housed in groups of 2 females and 1 male. Female rats were fed low-fluoride NIH-07 diet (7.95ppm fluoride). The diet was identical to that used in the NTP (1990) study obtained from Ziegler Bros. Inc., PA, USA.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Weight/volume NaF solutions were prepared in Aqua Cool Ultra Pure water (Ionics, Inc., MA, USA). Test solutions were presented to rats as drinking water available ad libitum. Only female rats were exposed to NaF drinking water (males were used as sires only).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No information available

Details on mating procedure:

Rats were cohabited from approximately 16.30hr on each mating day, until the following morning. The ratio of females to males was 2:1. On each morning after cohabitation, the females were removed from the mating cages and individually smeared for the presence of sperm in the vaginal lavage. The females that were confirmed as having mated were presumed pregnant and proceeded onto the study.

Duration of treatment / exposure:

20 days (from gestation days 0-20).

Frequency of treatment:

Daily

Duration of test:

From mating until gestation day 20.

No. of animals per sex per dose:

The numbers of females in each dose group (in ascending dose order): 35, 35, 35, 36, 37 and 37.

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were selected on the basis of doses used in the NTP carcinogenicity study (1990), with an additional lower and higher dose to increase the range. Once mating was confirmed, female rats were assigned to treatment groups by stratified random procedure.

Maternal examinations:

Fluid consumption was measured every 3 days, feed consumption was measured on gestation days 7, 14 and 20. Body weights were recorded at 3 day intervals during gestation and on day 20 prior to sacrifice. Behavioural signs and clinical toxicity were recorded. The numbers of corpora lutea were counted and recorded.

Ovaries and uterine content:

Caesarean sections were performed on gestation day 20. Each uterus was examined in situ for the presence and position of resorption sites, implantation sites, and live or dead foetuses. Deciduomas were called early deaths, and implantation sites with placentas and with complete but non-viable foetuses that were of subnormal size, that showed retarded development, or that were in a macerated condition, were classed as late deaths.

Fetal examinations:

Each viable foetus was weighed, sexed, measured for crown-rump length, and examined under magnification for the presence of external abnormalities. Viable foetuses were alternately evaluated for skeletal abnormalities using Alzarin Red S stain or for soft tissue abnormalities following serial sections.

Statistics:

ANOVA and two-tailed LSD test.

Indices:

Average percentage early + late deaths/litter

Historical control data:

No information

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was no dose-related behavioural changes or clinical signs. Water consumption was significantly reduced at 175 and 200 pm, and feed consumption was significantly reduced at 250 ppm, body weights reflected feed consumption trends; significant decreases in body weight gain were seen in 250 ppm females on days 0-3 and 6-9, and overall on days 0-20. The mean number of implants per litter was significantly reduced in the 250 ppm was significantly decreased, however findings correspond with a lower number of corpora lutea in this group.

Dose descriptor:

NOAEL

Effect level:

175 ppm (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
A significant increase was seen in the average number of foetuses with 3 or more skeletal variations in the 250 ppm group, and the numbers of litters with foetuses with 3 or more skeletal variations was also increased in this group but not significantly so.

Dose descriptor:

NOAEL

Effect level:

250 ppm (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

no

Water consumption by females was lower at dose levels of 175 and 200 ppm. This resulted in a lower than expected NaF consumption. Pregnancy rate was over 90% in all groups. A significantly lower number of corpora lutea was seen in the dams of the 250 ppm group. Implantation efficiency (% corpora lutea that implanted) was more than 90% in all groups except the 25 ppm group, although the small decrease was not significant. The occurrence of in utero deaths was similar in the control and treated groups. The mean number of male foetuses per litter was significantly decreased in the 175 ppm group compared to the control, but was not dose related therefore considered to be random.

Foetal growth was not affected by NaF, even in the high dose groups with dams exhibiting reduced food and water consumption. Male control foetuses weighed 4.0 g, and crown-rump length was 4.1cm; male foetuses from treated groups weighed 3.9 -4.1 g and crown-rump length was 4.0 -4.1 cm. Female control foetuses weighed 3.8 g and crown-rump length was 4.0 cm; female foetuses from treated groups weighed 3.7 -3.8 g and crown-rump length was 3.9 -4.0 cm.

Conclusions:

No evidence of developmental toxicity was seen in this study.

Executive summary:

The developmental toxicity of sodium fluoride was determined in rats. Mated females were exposed to sodium fluoride in the drinking water at concentrations of 0, 25, 100, 175 and 250 ppm on gestation days 0 -20. Caesarean sections were performed on gestation day 20 and foetuses were examined. Sodium fluoride was not teratogenic at any dose tested. There was no effect on the development of specific bones including sternebrae. Foetal growth was not affected by sodium fluoride, even in dams exhibiting significantly decreased food and water consumption (250 ppm; decreased feed and water consumption, 175 ppm decreased water consumption). A significant increase was seen in the average number of foetuses with three or more skeletal variations in the 250 ppm group, however the number of affected litters was not significantly increased. There was no dose related effect on sodium fluoride on the incidence of soft tissue variations.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-11-24 to 2013-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2001-01-22

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-07-09

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS- RccHan: WIST strain
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: approx. 12 weeks of age
- Weight at study initiation: control group: 197.9 - 230.9 g; low dose group: 198.1 - 230.2 g; mid dose group: 190.9 - 232.5 g; high dose group: 191.6 - 239.4 g
- Housing: animals were housed in Macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the quarantine and acclimatization periods, the animals were housed in groups of 4 per sex. Mated females were housed individually in Macrolon cages.
- Diet (ad libitum): cereal-based (closed formula) rodent diet (Rat & Mouse No.3 Breeding Diet; RM3) (supplier: SDS Special Diets Services, Witham, England)
- Water (ad libitum): domestic mains tap-water
- Quarantine period: 9 days (upon arrival the rats were quarantined and checked for overt signs of ill health and abnormalities. During the quarantine period, serological examinations of the microbiological status of the rats were conducted in a random sample.)
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Relative humidity: exceeded 65% for short times only during cleaning activities
- Air changes: about 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: demineralized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item in the vehicle were prepared weekly, and stored in a refrigerator (2-10°C) in the dark in aliquots sufficient for one day. The vehicle for dosing the controls was similarly stored.
The solutions of the test item in the vehicle were prepared by stirring on a magnetic stirrer. Subsequently, 8 aliquots (7 days plus 1 extra) were taken per dose level according to the daily volume required for each dosing. These aliquots were taken under continuous stirring. On each subsequent day, one aliquot for each group was removed from the refrigerator and allowed to equilibrate to ambient temperature prior to dosing.

A dosing volume of 10 mL/kg was applied for all animals, which was adjusted based on the latest body weight. After gestation day 14 dose volumes were not adjusted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From all three batches of the test items prepared in the study, samples were taken immediately after preparation and stored in a refrigerator until analysis. The following analyses were conducted by Inductively coupled plasma atomic emission spectrometry (ICP-AES) analysis. The test item was quantified using barium as a marker component:
- Homogeneity: the homogeneity of the test substance in the experimental test items was demonstrated in the first batch prepared, by analysing three samples (taken at different locations in the gavage liquid container) of each level.
- Concentration: the concentration of test substance at each level was determined in all three batches of test items prepared in the study.
- Stability: samples of the low-dose, mid-dose and high-dose level were analysed in the first batch prepared in the study at t=0 and after storage in the refrigerator (2 – 10 °C) for twelve days.

Results:
- Homogeneity: the relative standard deviations between the mean content at three different locations was < 5% in the low, mid and high dose level. Therefore barium chloride dihydrate was considered to be homogeneously distributed in each test.
- Stability: upon storage at refrigerator temperature from 22 November 2013 till 4 December 2013, the relative difference in test substance concentration between t=0 and t=4 days was -3.6, +1.5 and +4.2% in the low, mid and high dose level, respectively. And all the dose levels met the criteria for stability (relative difference ≤10%). Therefore it was concluded that there was no loss of test substance from any tests items during storage for twelve days in the refrigerator.
- Content: the content of barium chloride dihydrate determined in the test items are compared with the intended content. The relative difference between the mean determined content and the intended content was between 1.5 and 2.5% at all nominal levels of 1, 3 and 10 mg/ml which was within the acceptance criteria (relative difference ≤10%). Therefore, the actual content was considered to meet the intended level in each test item.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 2 females : one male
- Length of cohabitation: until a sperm positive smear was detected
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy

Duration of treatment / exposure:

gestation day 0 up to and including gestation day 20

Frequency of treatment:

daily

Duration of test:

25 days

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

24 mated female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels have been selected in consultation with the study monitor on the basis of a dose range finding study with the test item in pregnant rats.
During the dose range finding study groups of 5 mated females, were administered different dose levels of the test substance by gavage from gestation day 0 up to gestation day 21. A dose volume of 10 mL/kg body weight was applied and demineralized water was used as vehicle and control item. Dose levels of 0, 50, 175 and 250 mg/kg were administered.
Based on the preterm death of 3/5 females in the high dose group and 2/5 females in the mid dose group after a single dose, dosing was discontinued in both groups.
All surviving animals in the mid and high dose group were re-allocated to a new mid dose group and received 100 mg/kg body weight barium chloride from gestation day 2 onwards.
On gestation day 21 the animals were sacrificed and caesarean section was performed.
In-life parameters included clinical signs, morbidity, mortality, body weight and food consumption. At sacrifice uterus weight, number of corpora lutea, number of implantation sites, early and late resorptions, number of live and dead foetuses and foetus weight were recorded. In addition, foetuses were examined for external abnormalities/malformations and dams were observed for gross anatomical changes.

Results:
Oral administration of 0, 50, 100, 175 and 250 mg/kg barium chloride to mated females resulted in:
- the preterm death of 3/5 animals in the 250 mg/kg group and 2/5 animals in the 175 mg/kg group after a single oral dose.
- the spontaneous death of one animal in the 100 mg/kg group on gestation day 21. This animal was found dead before cesarean section and had 11 dead foetuses. This animal had received one dose of 250 mg/kg on gestation day 0 and daily doses of 100 mg/kg from gestation day 2 to 21.
- limited clinical observations in the 250 and 175 mg/kg group, including hunched posture an piloerection.
- no effect on body weight or body weight gain, food consumption, mean number of corpora lutea, implantation sites, early and late resorptions and the mean number of live foetuses.
- although based on a limited number of litters (four in the 50 mg/kg group and three in the 100 mg/kg group) an effect on foetus weight could not be ruled out.
- no foetuses showing external malformations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days (GD) 0, 3, 6, 10, 14, 17 and 21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (gestation days 0-3, 3-6, 6-10, 10-14, 14-17 and 17-21)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
The females were killed by decapitation after CO2/O2 anaesthesia on gestation day 21 and examined for gross abnormalities.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Weight of empty uterus: Yes
- Weight of ovaries: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Gross evaluation of placentas: Yes
For seemingly non pregnant females (part of) the uterus was stained with Na2SO3 in order to visualize possible implantation sites (Salewski E, 1964). Upon staining non pregnancy was confirmed for these females.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Further examinations:
- number of live and dead foetuses
- sex of the foetuses
- live foetuses (individually) and corresponding placentas
- foetal weight

Statistics:

Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 or p<0.01.
Continuous data were subjected to the ‘Decision tree for continuous data’ and dichotomous data to the ‘Decision tree for dichotomous data’.

Indices:

For each group the following indices were calculated:
- female fertility index = (no. of pregnant females/no. of inseminated females) x 100
- pre-implantation loss = [(no. of corpora lutea – no. of implantation sites) / no. of corpora lutea] x 100
- post-implantation loss = [(no. of implantation sites – no. of live foetuses) / No. of implantation sites] x 100
- gestation index = (no. of females with live foetuses/no. of females pregnant) x 100
- sex ratio = (no. of live male foetuses/no. of live foetuses) x 100

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: treatment-related death

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITIES
- two animals in the high dose group were found dead on day 21 of gestation. Both animals were pregnant and all foetuses were dead. Although death was not preceded by clinical signs, growth retardation or gross anatomical observations at necropsy that could clarify the death of these animals, the death of these rats is ascribed to treatment.
- one animal in the high dose group felt cold and was weakened and showed piloerection on gestation day 21. Upon necropsy this animal showed hydrothorax, haemorrhages in the liver and haemorrhagic discharge in the vagina. Also the death of this high-dose rat is ascribed to treatment.
- the spontaneous death of two rats, and the conditional decline of one rat on day 21 of gestation were considered to be treatment-related and to represent severe maternal toxicity in the high dose group.
- all foetuses were dead in the above three rats. The foetal deaths observed in these animals are considered to be related to the severe maternal toxicity in the high-dose group.

BODY WEIGHT AND BODY WEIGHT CHANGE
- no effects were observed on body weight. A slightly, but statistically significantly reduced body weight gain was observed in the high dose group as compared to the control group during the first three days of dosing. This was considered to be related to treatment and recovered thereafter.
- no effects on body weight or body weight gain were observed in the low dose group and the mid dose group as compared to the control group.

FOOD CONSUMPTION
- no effects were observed on food consumption.

REPRODUCTIVE PERFORMANCE
- 23, 22, 23 and 22 pregnant females in the control group, low dose, mid dose and high dose group, respectively.
- reproduction indices were comparable for the control, low dose, mid dose and high dose group
- no effects were noted in mean number of corpora lutea, mean number of implantation sites, preimplantation loss, mean number of early resorptions, late resorptions and mean number of live foetuses.

MACROSCOPY
- no treatment-related effects were observed

FEMALE REPRODUCTIVE ORGANS
- mean ovary weight, mean full and empty uterus weight were comparable in all groups
- mean carcass weight and net body weight change were comparable in all groups
- mean placenta weight was comparable in all groups

Dose descriptor:

NOAEL

Remarks:

(barium chloride dihydrate)

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Remarks:

(barium chloride)

Effect level:

25.6 mg/kg bw/day

Based on:

other: bariumdichloride

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FOETUS WEIGHT AND SEX
- mean foetus weight was comparable in all groups
- mean percentages male littermates was comparable in all groups

FOETAL EXAMINATION
- foetal external, visceral, and skeletal examinations did not reveal any treatment-related effects.

Dose descriptor:

NOAEL

Remarks:

(barium chloride dihydrate)

Effect level:

>= 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No effects observed at the highest dose level

Dose descriptor:

NOAEL

Remarks:

(barium chloride)

Effect level:

>= 85.3 mg/kg bw/day

Based on:

other: barium chloride

Basis for effect level:

other: No effects observed at the highest dose level

Remarks on result:

other: NOAEL calculated for barium chloride from the barium chloride dihydrate data.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Daily administration of barium chloride dihydrate at dose levels of 0, 10, 30 or 100 mg/kg body weight to pregnant rats from gestation day 1 up to and including gestation day 20, resulted in maternal toxicity as evidenced by the spontaneous deaths of two animals on gestation day 21 and the conditional decline of another animal on gestation day 21 in the high dose group. No developmental toxicity was observed.
The NOAEL for maternal toxicity was therefore 30 mg/kg body weight (recalculated for barium chloride: 25.6 mg/kg bw/day) . In absence of developmental effects the NOAEL for prenatal developmental toxicity in the rat was ≥ 100 mg/kg body weight (recalculated for barium chloride: ≥ 85.3 mg/kg bw/day).

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP study, only abstract available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Female New Zealand White Rabbits were fed standard laboratory chow ad libitum. Water (control and treated) was provided ad libitum.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

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

Analytical verification of doses or concentrations:

not specified

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:

Rabbits were mated, and dosing began on gestation day 6. No further information is available.

Duration of treatment / exposure:

Treatment from gestation day 6 to gestation day 19.

Frequency of treatment:

Daily

Duration of test:

30 days, beginning on gestation day 0.

No. of animals per sex per dose:

26 female rabbits per dose

Control animals:

yes, concurrent vehicle

Details on study design:

No further information.

Maternal examinations:

Animals were observed daily for clinical signs of toxicity. Food, water, and body weights were recorded for the animals in each group on gestation day 0 and every two days thereafter through gestation day 30. Blood samples were collected from 5 animals per group per replicate on gestation day 20; serum was delivered to the sponsor for determination of fluoride concentration. All animals were killed on gestation day 30 and examined for maternal body and organ weights, implant status, foetal weight, sex, and morphological development.

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:

No further information

Indices:

No further information

Historical control data:

No further information

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal toxicity occurred. Pregnancy rates were 84%, 87%, 78%, and 83% in the control to high exposure groups, respectively. Maternal body weight change for the animals receiving 400 ppm NaF was significantly lower than that of control animals for the period from gestation day 6 to 8 (14 grams average weight gain for controls vs. 112 grams weight loss for the 400 ppm group); this difference probably resulted from significantly decreased food and water consumption during the same period. Maternal body weight change was significantly increased from gestation day 10 to 12 (22 grams average weight gain for controls vs. 71 grams weight gain for the 400 ppm group), but did not differ among groups for the treatment period as a whole, indicating that animals in the 400 ppm group recovered from the weight change effects observed during the first few days of exposure to NaF in the drinking water. Maternal water consumption (g/kg/day) during exposure was significantly decreased in the animals exposed to 400 ppm NaF. Post-exposure water consumption was normal in these animals indicating the probability of dereased palatability of the 400 ppm solution. Maternal food consumption was decreased compared to control during the first four days of treatment (g/day on gestation day 6 to 8 and 8 to 10; g/kg/day on gestation day 6 to 8), but was normal thereafter. No clear clinical signs of toxicity were observed. Necropsy of the maternal animals revealed no effects on kidney or liver weights.

Dose descriptor:

NOAEL

Effect level:

200 ppm (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Exposure did not affect the frequency of post-implantation loss, mean foetal body weight per litter, or external, visceral, or skeletal malformations.

Dose descriptor:

NOAEL

Effect level:

400 ppm (nominal)

Basis for effect level:

other: No effects were seen at the highest dose level of 400 ppm

Abnormalities:

not specified

Developmental effects observed:

not specified

Drinking water contained less than 0.6 ppm of sodium fluoride (the detectable limit). Water intake provided approximately 84%, 91 % and 95% of the total F consumed for the low through high concentration groups in this study. Determination of serum fluoride levels, in 7-8 pregnant animals per group, revealed levels of 0.06 ± 0.04, 0.24 ± 0.10, 0.39 ± 0.14, and 0.70 ± 0.33 ppm at the end of the exposure period for the control through high dose groups, respectively.

Conclusions:

There was evidence of minimal maternal toxicity but no evidence of developmental toxicity with levels of sodium fluoride in drinking water as high as 400 ppm (resulting in an average exposure of 29 mg/kg bw/d) although the palatabillity of a 400 ppm sodium fluoride solution apparently reduced water consumption.

Executive summary:

Pregnant New Zealand White rabbits were exposed to sodium fluoride in their drinking water at concentrations of 0, 100, 200 or 400 ppm daily between gestation days 6 and 19. There was evidence of minimal maternal toxicity but no definitive evidence of developmental toxicity with levels of sodium fluoride in drinking water as high as 400ppm (resulting in an average exposure of 29 mg/kg bw/d) although the palatabillity of a 400 ppm sodium fluoride solution apparently reduced water consumption. This study established a NOAEL for maternal toxicity at 200 ppm NaF in drinking water (approximately 18 mg/kg bw/d) and a NOAEL for developmental toxicity of 400ppm NaF in drinking water (approximately 29 mg/kg bw/d) administered to pregnant NZW rabbits during organogenesis.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP study: abstract available

Qualifier:

according to guideline

Guideline:

other: NTP protocol

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Developmental toxicity study

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Sprague-Dawley CD rats were fed standard laboratory chow ad libitum. Water (control and treated) was provided ad libitum.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

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

Analytical verification of doses or concentrations:

not specified

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:

No further information is available; assumed pregnant females were dosed

Duration of treatment / exposure:

Treatment from gestation day 6 to gestation day 15.

Frequency of treatment:

Daily

Duration of test:

Animals were treated on Day6-15 of gestation and sacrific

No. of animals per sex per dose:

26 female rats per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

No further information

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, 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:

No further information

Indices:

No further information

Historical control data:

No further information

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal lethality occurred at any dose. Maternal wight gain was significantly reduced at 300ppm during the first 2 days of exposure (gestation days 6-8), and a trend toward decreased weight gain was noted for the treatment period as a whole. Maternal food intake was significantly decreased (compared to controls) in the 300 ppm group between gestation days 8-10. Water consumption was significantly decreased during exposure in the 300 ppm group. No other differences were noted. At necropsy there were no effects on kidney or liver weights.

Dose descriptor:

NOAEL

Effect level:

150 ppm (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
NaF exposure did not significantly affect the frequency of post-implantation loss, mean fetal body weight per litter, or external, visceral, or skeletal malformations.

Dose descriptor:

NOAEL

Effect level:

300 ppm (nominal)

Basis for effect level:

other: There was no evidence of developmental toxicity in this study at the highest dose level tested

Remarks on result:

other: 27 mg NaF/kg/day

Abnormalities:

not specified

Developmental effects observed:

not specified

- 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.

Conclusions:

Sodium fluoride in drinking water was not maternally toxic up to doses of 300ppm, although decreased water consumption was seen as a result of poor palatability at this dose. There was no evidence of developmental toxicity in this study.

Executive summary:

Pregnant Sprague-Dawley CD rats were exposed to sodium fluoride in their drinking water at concentrations of 0, 50, 150 or 300 ppm daily between gestation days 6 and 15. Maternal weight gain was significantly reduced at 300 ppm during the first two days of exposure (days 6 to 16). Maternal water consumption (grams/kg/day) during exposure was significantly decreased in the animals exposed to 300 ppm NaF. Post-exposure water consumption was normal in these animals indicating the probability of decreased palatability of the 300 ppm solution. Necropsy of the maternal animals revealed no effects on kidney or liver weights. NaF exposure did not significantly affect the frequency of post-implantation loss, mean fetal body weight per litter, or external, visceral, or skeletal malformations.

This study established a NOAEL for maternal toxicity of 150 ppm (18 mg NaF/kg bw/d) and a NOAEL of 300 ppm for developmental toxicity (27 mg NaF/kg bw/d) administered in drinking water to pregnant CD rats during organogenesis.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

51.3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No developmental toxicity study is available on barium fluoride and therefore this endpoint was assessed in a "Weight-of-Evidence" approach in accordance with section 1.2 of REACH Annex XI. Under physiological conditions barium fluoride will dissociate into barium and fluoride ions. Therefore the toxicity of barium fluoride may reasonably be considered to be determined by the availability of Ba2+ cations and F- anions. Comprehensive data on developmental toxicity are available for barium chloride and sodium fluoride. As a first surrogate for bioavailability, the water solubility of a test substance may be used. Both barium chloride and sodium fluoride are highly water soluble with ca. 375 g/L and 40 g/L, respectively at neutral pH, whereas barium fluoride has a solubility of 1.6 g/L. Hence, any read across from barium chloride and sodium fluoride to BaF2 is inherently conservative.

Fluoride

In a rat developmental toxicity study (NTP, 1994; Heindel et al, 1996), maternal toxicity (transiently reduced bodyweight gain) was apparent at the highest dose level of 300 ppm sodium fluoride (in drinking water), equivalent to 13 mg/kg bw/d fluoride. No evidence of developmental toxicity was seen at this dose level. No clear evidence of developmental toxicity was seen in an FDA rat study (Collins et al,1995) at dose levels of up to 250 ppm sodium fluoride in drinking water (equivalent to 12.3 mg/kg bw/d fluoride). Maternal toxicity in this study was limited to reduced food intake at the highest dose level. No evidence of developmental toxicity was seen in a rabbit study (NTP, 1993; Heindel et al, 1996) at dose levels of up to 400 ppm sodium fluoride (equivalent to 14 mg/kg bw/d fluoride from all sources)

Barium

Developmental toxicity of barium chloride dihydrate was evaluated in a prenatal developmental toxicity study by daily administration of the test item at dose levels of 0, 10, 30 or 100 mg BaCl2 * 2 H2O/kg body weight to pregnant rats from gestation day 1 up to and including gestation day 20. No effects on body weights, food consumption and clinical signs were observed. Maternal toxicity was evidenced by the spontaneous deaths of two animals on gestation day 21 only and the conditional decline of another animal on gestation day 21 in the high dose group. No developmental toxicity or treatment-related observations, whatsoever in external, visceral and skeletal foetal examinations were observed in any dose level. The NOAEL for maternal toxicity was therefore 30 mg/kg body weight barium chloride dihydrate (16.9 mg Ba2 +/kg bw/d). In absence of developmental effects, the NOAEL for prenatal developmental toxicity in the rat was ≥ 100 mg/kg body weight barium chloride dihydrate (corresponding to 80.7 mg Ba2 +/kg bw/d).

Justification for classification or non-classification

Reliable studies do not indicate any developmental toxicity or reproductive toxicity of barium fluoride. In accordance to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for effects on fertility and development.