Fluorine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

From the two-generation study by Collins et al. (2001) a NOAEL for reproductive performance of 250 ppm in drinking water can be derived (highest dose tested).

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 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

From a two-generation study by Collins et al. (2001a) 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 b.w./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 b.w./d or 12.5 mg F-/kg b.w./d for males and non-pregnant females respectively.

Effects on developmental toxicity

Description of key information

From three well-performed embryo- and developmental toxicity studies with NaF an overall NOAEL for maternal toxicity and developmental effects of 11.12 mg F-/kg b.w./d can be derived.

Link to relevant study records

Reference

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

11.12 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

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 (Collinset 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).

Justification for classification or non-classification

Reliable studies do not indicate any developmental toxicity or reproductive toxicity of fluoride. No classification is therefore proposed for fluorine.