Disodium fluorophosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was performed between 06 May 2010 and 19 October 2010. The in-life phase of the study was conducted between 11 May 2010 (first day of treatment) and 28 June 2010 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to GLP and guideline. This study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement for this endpoint as a part of a weight of evidence approach in accordance with Annex XI of Regulation EC No. 1907/2006.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (approximately twelve weeks old
- Weight at study initiation: males weighed 307 to 370g, the females weighed 189 to 232g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Humidity: 55 ± 15%
- Air changes: at least fifteen air changes per hour
- Photoperiod: 12hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Distilled water

VEHICLE
- Concentration in vehicle: 0, 6, 14 and 30 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Details on mating procedure:

- M/F ratio per cage: 1 male: 1 female basis within each dose group
- Length of cohabitation: up to fourteen days
- Proof of pregnancy Each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)
- After successful mating each pregnant female was housed individually during the period of gestation and lactation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Disodium fluorophosphate in the test item formulations was determined by ion chromatography (IC) using an external standard technique. The results indicate that the prepared formulations were within ± 6% of the nominal concentration (see table 1).

Duration of treatment / exposure:

Up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 70 and 150 mg/kg/day

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 30, 70, 150 mg/kg/day
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.


ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.


ix) Blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing soon after dosing, and one hour after dosing at weekends and public holidays

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Measured daily throughout the study


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 post partum for females
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
- Parameters examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), Platelet count (PLT), Reticulocyte count (Retic) Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 post partum for females
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
- Parameters examined: Urea Calcium (Ca++), Glucose Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids (Bile)


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to termination
- Dose groups that were examined: five selected males and females from each dose level
- Battery of functions tested: sensory activity, grip strength and motor activity

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live and dead births, weight gain, clinical condition, surface righting reflex

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were terminated on Day 43
- Maternal animals: All surviving animals females were terminated on day 5 post partum


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were tissues were prepared for microscopic examination:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Oesophagus
Caecum Rectum
Coagulating gland Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides Skin (hind limb)
Eyes* Spinal cord (cervical, mid-thoracic and
Gross lesions lumbar)
Heart Spleen
Ileum (including peyers patches) Stomach
Jejunum Thyroid/parathyroid
Kidneys Trachea
Liver Testes
Lungs (with bronchi)
Thymus
Lymph nodes (cervical and mesenteric) Urinary bladder
Mammary tissue Uterus/Cervix
Muscle (skeletal) Vagina

The following organs, removed from animals that were killed at the end of the study:
Adrenals Prostate
Brain Seminal vesicles
Epididymides Spleen
Heart Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix)
Pituitary

Statistics:

Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test. Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number of animals mated /Number of animals paired x 100

Pregnancy Index (%) = Number of pregnant females/Number of animals mated x 100

Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation

i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = Number of females delivering live offspring/Number of pregnant females x 100

Offspring viability indices:

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss = Number of corpora lutea - number of implantation sites/number of corpora lutea x 100
Post–implantation loss = Number of implantation sites - Total number of offspring born / Number of implantation sites x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = Number of offspring alive on Day 1/Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4/ Number of offspring alive on Day 1 x 100

iii) Sex Ratio (% males) calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Number of male offspring/Total number of offspring x 100

Clinical signs:

effects observed, treatment-related

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male treated with 150 mg/kg/day was killed in extremis on Day 7. Two females from this treatment group were killed in extremis prior to littering on Day 42 and a further female from this treatment group was killed in extremis prior to littering on Day 49. The cause of the moribund condition could not be established histopathologically There were no further unscheduled deaths.

Increased salivation was detected in animals of either sex treated with 150 mg/kg/day from Day 9 (males) and Day 22 (females) onwards. An isolated incident of staining around the mouth was evident in one male treated with 150 mg/kg/day on Day 15 and staining around the snout was also evident in two males from this treatment group between Days 29 and 32. Observations at 70 mg/kg/day were confined to increased salivation in males from Day 15 onwards, staining around the mouth in two males on Days 29 and 30 and noisy respiration in one male and one female between Days 30 and 42. The animals treated with 150 mg/kg/day that were killed in extremis showed either a decreased respiratory rate, ptosis, pilo-erection, lethargy, hypothermia, hunched posture dehydration, fur staining, pallor of the extremities, tiptoe gait and/or increased salivation prior to termination. No such effects were detected in animals of either sex treated with 30 mg/kg/day.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Animals of either sex treated with 150 mg/kg/day showed a reduction in body weight gain during the first week of maturation with actual body weight losses being evident in four males and six females. Recovery in body weight gain was evident in males throughout the remaining treatment period however a reduction in body weight gain was evident in females from this treatment group during the final week of gestation and during lactation. Three females treated with 150 mg/kg/day showed actual body weight losses during lactation. Statistical analysis did not however reveal any significant intergroup differences during maturation, gestation or lactation. No such effects were detected in animals of either sex treated with 70 or 30 mg/kg/day. Males treated with 150 mg/kg/day showed a statistically significant increase in body weight gain during the final week of treatment however an increase in body weight gain is considered not to be of toxicological significance.
No adverse effect on food consumption was detected for males during the treatment period or for females during the pre-mating, gestation or lactation phases of the study
Food efficiency was however reduced during the first week of treatment for animals of either sex treated with 150 mg/kg/day. Food efficiency for these animals was comparable to controls thereafter.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating There were no treatment-related effects on mating for treated animals. One control female was found to be non pregnant.

Fertility. There were no treatment-related effects on conception rates for treated animals. One control female failed to show positive evidence of mating but subsequently gave birth to live offspring.

Gestation Lengths. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Animals of either sex treated with 150 mg/kg/day showed a statistically significant increase in kidney weight (p<0.05-0.01) both absolute and relative to terminal body weight. Males treated with 150 mg/kg/day also showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. No toxicologically significant effects were detected in animals of either sex treated with 70 or 30 mg/kg/day. Females from all treatment groups showed statistically significant reductions in thymus weight and statistically significant increases in spleen weight both absolute and relative
to terminal body weight. Males treated with 150 and 70 mg/kg/day showed statistically significant reductions in prostate weight both absolute and relative to terminal body weight. In the absence of a true dose related response or any histology correlates the intergroup differences were considered to be of no toxicological significance. Females treated with 150 mg/kg/day showed a statistically significant increase in ovary weight both absolute and relative to terminal body weight. Males treated with 150 mg/kg/day showed a statistically significant reduction in absolute and relative semincal vesicle weight and a statistically significant increase in absolute and relative brain weight. Males treated with 70 mg/kg/day also showed a statistically significant increase in absolute and relative brain weight whilst males treated with 30 mg/kg/day showed a statistically significant reduction in absolute and relative thyroid/parathyroid weight. In the absence of any histology correlates the intergroup differences were considered to be of no toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Five males treated with 150 mg/kg/day showed a raised limiting ridge at necropsy and a thickened glandular region of the stomach. In addition, one male also showed enlarged cervical lymph nodes, one male also showed small seminal vesicles, pale and enlarged kidneys and pale adrenals and two males also showed a reddened glandular region of the stomach. A further male treated with 150 mg/kg/day showed pale adrenals and enlarged, pale and mottled kidneys. One female treated with 150 mg/kg/day showed pale adrenals and enlarged and pale kidneys. The male treated with 150 mg/kg/day that was killed in extremis showed a raised limiting ridge, reddened lungs, enlarged and pale kidneys together with hydronephrosis and a dark liver. The females from this treatment group that were killed in extremis showed enlarged and pale adrenals, pale and/or enlarged kidneys, either a pale liver, pale and reddened lungs, dark red contents in the stomach, a distended stomach, a raised limiting ridge, a pale glandular and/or non glandular region of the stomach or a red duodenum.ridge, a pale glandular and/or non glandular region of the stomach or a red duodenum. Two females treated with 70 mg/kg/day had sloughing on the glandular region of the
stomach. One of these females also showed enlarged lymph nodes. One female from this treatment group had a mottled liver and dark foci on the lungs and a further female from this treatment group had hydronephrosis in the right kidney. Seven males treated with 70 mg/kg/day and three males treated with 30 mg/kg/day showed a raised limiting ridge of the stomach at necropsy. One of these males from each treatment group also showed a thickened glandular region of the stomach. One control male had hydronephrosis in the right kidney and a further control male had a mass on the caudate lobe of the liver. In the absence of treatment these findings were of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The following treatment-related microscopic findings were detected:
Stomach: Hyperkeratosis and acanthosis, mainly at the limiting ridge, was evident in animals of either sex from all treatment groups. Parietal cell hypertrophy in the glandular mucosa was evident in animals of either sex treated with 150 and 70 mg/kg/day and in males treated with 30 mg/kg/day. Submucosal inflammation was also noted in males from all treatment groups. Glandular erosion of the stomach was noted in two males treated with 150 mg/kg/day and two females from this treatment group also showed superficial desquamation of the glandular surface.

Kidneys: Tubular degeneration and tubular simple dilation was noted in animals of either sex treated with 150 mg/kg/day and in males treated with 70 mg/kg/day. Granulocytic casts and interstitial nephritis were also evident in animals of either sex treated with 150 mg/kg/day

Submaxillary Gland: Acinar cell atrophy was evident in four males and two females treated with 150 mg/kg/day and one female treated with 70 mg/kg/day.

Pancreas: Acinar cell atrophy was evident in one male and six females treated with 150 mg/kg/day and in one female treated with 70 mg/kg/day. Fibrosis was also noted in three females treated with 150 mg/kg/day.

Mammary Gland: Acinar atrophy was evident in six males treated with 150 mg/kg/day, two males treated with 70 mg/kg/day and one male treated with 30 mg/kg/day.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

duodenum

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
A slight reduction in offspring viability was evident in litters from females treated with 150 mg/kg/day. Statistical significance was not achieved in this parameter and the effect was considered to be a non specific effect that may be related to maternal toxicity at this dose level rather than a direct reproductive effect.

CLINICAL SIGNS (OFFSPRING)
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test material toxicity.

BODY WEIGHT (OFFSPRING)
There were no significant differences in litter weights. A slight reduction in offspring body weight gain was evident in litters from females treated with 150 mg/kg/day. Statistical significance was not achieved in this parameter and the effect was considered to be a non specific effect that may be related to maternal toxicity at this dose level rather than a direct reproductive effect.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

70 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Reproductive effects observed:

not specified

Table 1: Verification of Concentration of Test Item Formulations

Analysis number	Nominal concentration (mg/ml)	Concentration found
		(mg/ml)	(expressed as % of nominal)
1	0 6 14 30	ND 5.88 13.8 30.0	- 98 98 100
2	0 6 14 30	ND 5.72 14.1 29.8	- 95 101 99
3	0 6 14 30	ND 6.18 14.4 31.0	- 103 103 103
4	0 6 14 30	ND 5.62 14.7 30.0	- 94 105 100

Conclusions:

Non-statistically significant treatment-related effects were observed in offspring development and offspring viability in litters from females treated with 150 mg/kg/day. These effects were considered to be related to maternal toxicity. No such effects were detected in litters from females treated with 70 or 30 mg/kg/day, therefore, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 70 mg/kg/day.
This study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement for this endpoint as a part of a weight of evidence approach in accordance with Annex XI of Regulation EC No. 1907/2006.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

70 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Three studies are available and as such a weight of evidence is provided. The screening study has a Klimisch score of 1 and the literature data on NaF has a Klimisch score of 2. The overal quality of the database is considered to be high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for the choice of effect level to be used in the CSR

A weight of evidence approach was applied to the REACH endpoint 8.7 Reproductive toxicity (including 8.7.3. Two-generation reproductive toxicity study). Three studies have been reviewed; two studies on the analogous substance sodium fluoride (see below for justification) and one study on disodium fluorophosphate.

Collins et al (2001) reported that no adverse effects on reproduction, mating, fertility and survival indices were noted when sodium fluoride was administered in the drinking water of three generations of rats in concentrations up to 250 ppm (equivalent to 12.8 mg F/ kg bw/day). In addition, a study by Sprando et al (2007) looked specifically at the effects of sodium fluoride on the parameters of fertility in two generations of male Sprague-Dawley rats. Sprando concluded that there were no effects to the fertility, in particular, spermatogenesis, of the male rats when tested with concentrations up to 250 ppm (equivalent to 12.8 mg F/ kg bw/day). Both publications were based on the same set of results.

An OECD 422 (combined repeated dose and reproductive/developmental screening study) has been performed on the substance disodium fluorophosphate. This study showed non-statistically significant treatment-related effects on development and viability of offspring in the highest dose group (150 mg/kg bw/day). Specifically, a slight reduction in offspring numbers and decreased bodyweight gains and litter weights were observed in litters from females treated with 150 mg/kg bw/day disodium fluorophosphate. These effects although not significantly significant, were likely to be incidental and were attributed to the effects of maternal toxicity. No effects were noted at 30 mg/kg bw or 70 mg/kg bw.

The NOEL was therefore determined to be 70 mg/kg bw/day. On the basis of the above results this value has been chosen for use in the CSR as it is the lowest and most conservative value; the studies performed on sodium fluoride could be extrapolated to disodium fluorophosphate on the basis of a molecular weight calculation and would give an estimated NOAEL of ca. 98.5 mg/kg bw/day. 

Justification for read-across from sodium fluoride to sodium fluorophosphate:

Sodium fluoride (NaF) is considered to be analogous to disodium fluorophosphate in terms of toxicological profile for the following reasons;

- A combined 28-day reprotoxicity screening toxicity study (OECD 422) performed on disodium fluorophosphate confirmed the leading health effects to be consistent with the effects reported for exposure to inorganic fluorides (fluorosis). In particular severe effects were noted in the mucosal lining of the stomach and are consistent with exposure to fluoride.

- Results of studies on NaF are considered to present a worst case for exposure as NaF contains approximate 47% F compared to 13% F in disodium fluorophosphate.

- The phosphate content of sodium fluorophosphate is not considered to present a hazard in terms of reprotoxicity. Phosphate is an essential element; a maximum tolerable daily intake value (MTDI) for phosphates is reported to be 70 mg/kg bw/day. This value greatly exceeds the limit value placed on an inorganic fluoride (OEL = 2.5 mg/kg bw/day F) and as such is not considered to be relevant for risk assessment.

Short description of key information:
A weight of evidence approach has been applied to the endpoint ‘reprotoxicity’. One OECD 422 study (Dunster J, 2010) on disodium fluorophosphate exists. In addition two longer-term studies of the analogous substance sodium fluoride are available (Collins T, 2001 and Sprando RL, 1997). In accordance with Regulation (EC) No. 1907/2006, Annex XI, Section 1.2 further testing on vertebrates for this endpoint is considered to be scientifically unnecessary as the data are sufficient to draw conclusions on the reprotoxicity of disodium fluorophosphate.

Justification for selection of Effect on fertility via oral route:
Study provides the key value for risk assessment.

Effects on developmental toxicity

Description of key information

A weight of evidence approach has been applied to the endpoint ‘developmental toxicity’. One OECD 422 study (Dunster J, 2010) on disodium fluorophosphate exists. A Developmental toxicity study equivalent to OECD 414 is available for the analogous substance sodium fluoride (NTIS, 1994) and a number of publications describing a 3-generation study in rats are also available for sodium fluoride (Collins T, 2001, Collins T, 1995).  Finally, data on disodium hydrogenorthophosphate (CAS No. 7558-80-7) are submitted to support the conclusion that sodium and phosphate do not contribute to the developmental toxicity potential of disodium fluorophosphate.
In accordance with Regulation (EC) No. 1907/2006,  Annex XI, Section 1.2 further testing on vertebrates for this endpoint is considered to be scientifically unnecessary as the data are sufficient to draw conclusions on the developmental toxicity of disodium fluorophosphate.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was performed between 06 May 2010 and 19 October 2010. The in-life phase of the study was conducted between 11 May 2010 (first day of treatment) and 28 June 2010 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to GLP and guideline. This study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement for this endpoint as a part of a weight of evidence approach in accordance with Annex XI of Regulation EC No. 1907/2006.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (approximately twelve weeks old)
- Weight at study initiation: males weighed 307 to 370g, the females weighed 189 to 232g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Humidity: 55 ± 15%
- Air changes: at least fifteen air changes per hour
- Photoperiod: 12hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Distilled water

VEHICLE
- Concentration in vehicle: 0, 6, 14 and 30 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Disodium fluorophosphate in the test item formulations was determined by ion chromatography (IC) using an external standard technique. The results indicate that the prepared formulations were within ± 6% of the nominal concentration (see table 1).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male: 1 female basis within each dose group
- Length of cohabitation: up to fourteen days
- Proof of pregnancy Each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)
- After successful mating each pregnant female was housed individually during the period of gestation and lactation

Duration of treatment / exposure:

Up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 70 and 150 mg/kg/day

Frequency of treatment:

Daily

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.


ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.


ix) Blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing soon after dosing, and one hour after dosing at weekends and public holidays

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Measured daily throughout the study


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 post partum for females
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
- Parameters examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), Platelet count (PLT), Reticulocyte count (Retic) Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 post partum for females
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
- Parameters examined: Urea Calcium (Ca++), Glucose Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids (Bile)


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to termination
- Dose groups that were examined: five selected males and females from each dose level
- Battery of functions tested: sensory activity, grip strength and motor activity
SACRIFICE
- Male animals: All surviving animals were terminated on Day 43
- Maternal animals: All surviving animals females were terminated on day 5 post partum


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were tissues were prepared for microscopic examination:
Adrenals , Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skin (hind limb), Eyes, Spinal cord (cervical, mid-thoracic and Gross lesions lumbar), Heart , Spleen, Ileum (including peyers patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes, Lungs (with bronchi) , Thymus, Lymph nodes (cervical and mesenteric), Urinary, bladder, Mammary tissue, Uterus/Cervix, Muscle (skeletal), Vagina

The following organs, removed from animals that were killed at the end of the study:
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
On completion of parturition (Day 0 post-partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 postpartum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post-partum
iii) Sex of offspring on Days 1 and 4 post-partum.
iv) Clinical condition of offspring from birth to Day 5 post-partum.
v) Individual offspring weights on Days 1 and 4 post-partum (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on Day 1 post-partum.

Statistics:

Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test. Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Indices:

Offspring viability indices
 
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
 
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss = Number of corpora lutea - number of implantation sites/number of corpora lutea x 100
Post–implantation loss = Number of implantation sites - Total number of offspring born / Number of implantation sites x 100
 
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = Number of offspring alive on Day 1/Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4/ Number of offspring alive on Day 1 x 100
 
iii) Sex Ratio (% males) calculated for each litter value on Days 1 and 4 post-partum, using the following formula: Number of male offspring/Total number of offspring x 100

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
A slight reduction in offspring viability was evident in litters from females treated with 150 mg/kg/day. Statistical significance was not achieved in this parameter and the effect was considered to be a non specific effect that may be related to maternal toxicity at this dose level rather than a direct reproductive effect.

CLINICAL SIGNS (OFFSPRING)
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test material toxicity.

BODY WEIGHT (OFFSPRING)
There were no significant differences in litter weights. A slight reduction in offspring body weight gain was evident in litters from females treated with 150 mg/kg/day. Statistical significance was not achieved in this parameter and the effect was considered to be a non specific effect that may be related to maternal toxicity at this dose level rather than a direct reproductive effect.

Dose descriptor:

NOAEL

Effect level:

70 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Abnormalities:

not specified

Developmental effects observed:

not specified

Results.

Adult Responses:

Mortality. One male treated with 150 mg/kg/day was killed in extremis on Day 7. Two females from this treatment group were killed in extremis prior to littering on Day 42 and a further female from this treatment group was killed in extremis prior to littering on Day 49. There were no further unscheduled deaths.

Clinical Observations. Episodes of increased salivation, noisy respiration or fur staining were evident in animals of either sex treated with 150 and 70 mg/kg/day throughout the treatment period. The animals treated with 150 mg/kg/day that were killed in extremis showed either a decreased respiratory rate, ptosis, pilo-erection, lethargy, hypothermia, hunched posture, dehydration, fur staining, pallor of the extremities, tiptoe gait and/or increased salivation prior to termination. No such effects were detected in animals of either sex treated with 30 mg/kg/day.

Behavioural Assessment. There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests. There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments. There were no toxicologically significant changes in sensory reactivity.

Body Weight. Animals of either sex treated with 150 mg/kg/day showed a reduction in body weight gain during the first week of maturation with actual body weight losses being evident. Recovery in body weight gain was evident in males throughout the remaining treatment period. A reduction in body weight gain was evident in females from this treatment group during the final week of gestation and during lactation. Three females treated with 150 mg/kg/day showed actual body weight losses during lactation. No such effects were detected in animals of either sex treated with 70 or 30 mg/kg/day.

Food Consumption. No adverse effects on food consumption or food efficiency were detected.

Water Consumption. Animals of either sex treated with 150 mg/kg/day showed an increase in overall water consumption throughout the treatment period. No such effects were detected in animals of either sex treated with 70 or 30 mg/kg/day.

Reproductive Performance:

Mating: There were no treatment-related effects on mating for treated animals. One control female was found to be non-pregnant.

Fertility: There were no treatment-related effects on conception rates for treated animals. One control female failed to show positive evidence of mating but subsequently gave birth to live offspring.

Gestation Lengths: There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size and Viability: Litter size at birth and subsequently on Days 1 and 4 post-partum were reduced in females treated with 150 mg/kg/day. Statistical analysis did not however reveal any significant intergroup differences.

No such effects were detected in litters from females treated with 70 or 30 mg/kg/day.

Offspring Growth and Development: Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post-partum were reduced in litters from females treated with 150 mg/kg/day. Viability index was also reduced in litters from this treatment group. Statistical analysis did not however reveal any significant intergroup differences.

Laboratory Investigations:

Haematology: Males treated with 150 mg/kg/day showed an increase in neutrophil count. No such effects were detected in females treated with 150 mg/kg/day or animals of either sex treated with 70 or 30 mg/kg/day.

Blood Chemistry: Animals of either sex treated with 150 mg/kg/day showed a reduction in plasma chloride concentration and an increase in inorganic phosphorus. Males treated with 150 mg/kg/day also showed an increase in calcium concentration, cholesterol and bilirubin whilst females from this treatment group also showed an increase in urea, aspartate aminotransferase, alanine aminotransferase and creatinine. The effect on alanine aminotransferase also extended to 70 mg/kg/day females. No toxicologically significant effects were detected in males treated with 70 mg/kg/day or in animals of either sex treated with 30 mg/kg/day.

Pathology:

Organ Weights: Animals of either sex treated with 150 mg/kg/day showed an increase in kidney weight both absolute and relative to terminal body weight. Males treated with 150 mg/kg/day also showed an increase in absolute and relative liver weight. No such effects were detected in animals of either sex treated with 70 or 30 mg/kg/day.

Necropsy: Macroscopic observations in terminal kill 150 mg/kg/day animals consisted of either a raised limiting ridge in the stomach, a thickened, pale or reddened glandular region of the stomach, enlarged cervical lymph nodes, small seminal vesicles, pale adrenals, enlarged, pale or mottled kidneys, a pale liver or a reddened duodenum. Similar effects albeit to a lesser extent were also evident at 70 and 30 mg/kg/day. The male treated with 150 mg/kg/day that was killed in extremis showed a raised limiting ridge, reddened lungs, enlarged and pale kidneys together with hydronephrosis and a dark liver. The females from this treatment group that were killed in extremis showed enlarged and pale adrenals, pale and/or enlarged kidneys, either a pale liver, pale and reddened lungs, dark contents in the stomach, a distended stomach, a raised limiting ridge, a pale glandular and/or non glandular region of the stomach or a red duodenum.

Histopathology: The following treatment-related microscopic findings were detected:

Stomach: Hyperkeratosis and acanthosis, mainly at the limiting ridge, was evident in animals of either sex from all treatment groups. Parietal cell hypertrophy in the glandular mucosa was evident in animals of either sex treated with 150 and 70 mg/kg/day and in males treated with 30 mg/kg/day. Submucosal inflammation was also noted in males from all treatment groups. Glandular stomach erosion was noted in two males treated with 150 mg/kg/day and two females from this treatment group also showed superficial desquamation of the glandular surface.

Kidneys: Tubular degeneration and tubular simple dilation was noted in animals of either sex treated with 150 mg/kg/day and in males treated with 70 mg/kg/day. Granulocytic casts and interstitial nephritis were also evident in animals of either sex treated with 150 mg/kg/day.

Submaxillary Gland: Acinar cell atrophy was evident in four males and two females treated with 150 mg/kg/day and one female treated with 70 mg/kg/day.

Pancreas: Acinar cell atrophy was evident in one male and six females treated with 150 mg/kg/day and in one female treated with 70 mg/kg/day. Fibrosis was also noted in three females treated with 150 mg/kg/day.

Mammary Gland: Acinar atrophy was evident in six males treated with 150 mg/kg/day, two males treated with 70 mg/kg/day and one male treated with 30 mg/kg/day.

Conclusions:

Treatment-related effects were observed in offspring development and offspring viability in litters from females treated with 150 mg/kg/day. No such effects were detected in litters from females treated with 70 or 30 mg/kg/day, therefore, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 70 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

70 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Five studies are available and as such a weight of evidence is provided. The screening study has a Klimisch score of 1 and the literature data on NaF has a Klimisch score of 2. The overal quality of the database is considered to be high.

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

Justification for the choice of effect level to be used in the CSR:

A weight of evidence approach was applied to the endpoint 8.7.2. Prenatal developmental toxicity. Four studies have been reviewed; three studies on the analogous substance sodium fluoride (see below for justification) and one study on disodium fluorophosphate.

An OECD 422 (combined repeated dose and reproductive/developmental screening study) has been performed on the substance disodium fluorophosphate. This study showed non-statistically significant treatment-related effects on development and viability of offspring in the highest dose group (150 mg/kg bw/day). Specifically, a slight reduction in offspring numbers and decreased bodyweight gains and litter weights were observed in litters from females treated with 150 mg/kg bw/day disodium fluorophosphate. These effects although not significantly significant, were likely to be incidental and were attributed to the effects of maternal toxicity. No effects were noted at 30 mg/kg bw or 70 mg/kg bw.

Developmental toxicity studies, equivalent to OECD 414, have been performed on New Zealand White rabbits and Sprague-Dawley rats with the test substance sodium fluoride (Heindel, 1993 and Heindel , 1994 respectively).

The studies, performed as part of the U.S. National Toxicology Program (NTP) concluded the following:

-          The NOAEC for developmental toxicity was determined to be 300 ppm sodium fluoride.    

- Collins et Al (1995) conducted a developmental toxicity study in which pregnant rats were given drinking water containing sodium fluoride at concentrations of 0, 10, 25, 100, 175 or 250 ppm throughout gestation. 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 250 ppm (25.1 mg/kg/day) were found. 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.   Maternal toxicity was also observed at 250 ppm; pregnant rats drank significantly less fluid and ate less food than did controls throughout gestation. These findings were reflected by significant decreases in body weight gain at 250 ppm.

Collins et al (2001) evaluated the developmental effects of continuous exposure to 0, 25, 100, 175 or 250 ppm sodium fluoride in drinking water over 3 generations of rats. A concentration of 175 ppm sodium fluoride in drinking water of a parental (F0) and two filial generations (F1 and F2) of rats did not produce developmental effects and is considered a no-observed effect concentration for developmental toxicity. A concentration of 250 ppm sodium fluoride in drinking water of a parental (F0) and two filial generations (F1 and F2) of rats caused statistically significant decrease in ossification of the hyoid bone in F2 foetuses and is considered an effect concentration for developmental toxicity.

A teratogenicity study on sodium dihydrogenorthophosphate (CAS number: 7558-80-7) is also included to support the conclusions that it is the F content of disodium fluorophosphate that is responsible for the leading health effects. Sodium phosphates are not considered to be developmental toxicants; this is supported by the study (Bailey, 1975) which details the developmental toxicity of sodium dihydrogenorthophosphate in rats and mice. No effects were noted in mice or rats up to the highest dose level tested. The NOAEL for developmental toxicity and maternal toxicity in rats was determined to be > 370 mg/kg bw. The NOAEL for developmental toxicity and maternal toxicity in mice was determined to be > 410 mg/kg bw.

 

The NOEL for the purpose of risk assessment has therefore been determined to be 70 mg/kg bw/day. On the basis of the above results this value has been chosen for use in the CSR as it is the lowest and most conservative value; the studies performed on sodium fluoride could be extrapolated to disodium fluorophosphate on the basis of a molecular weight calculation but would give higher values and would be less reliable.

Justification for read-across from sodium fluoride to sodium fluorophosphate:

Sodium fluoride (NaF) is considered to be analogous to disodium fluorophosphate in terms of toxicological profile for the following reasons;

- A combined 28-day reprotoxicity screening toxicity study (OECD 422) performed on disodium fluorophosphate confirmed the leading health effects to be consistent with the effects reported for exposure to inorganic fluorides (fluorosis). In particular severe effects were noted in the mucosal lining of the stomach and are consistent with exposure to fluoride.

- Results of studies on NaF are considered to present a worst case for exposure as NaF contains approximate 47% F compared to 13% F in disodium fluorophosphate.

- The phosphate content of sodium fluorophosphate is not considered to present a hazard in terms of developmental toxicity (see supporting data; Bailey, 1975). Phosphate is an essential element; a maximum tolerable daily intake value (MTDI) for phosphates is reported to be 70 mg/kg bw/day. This value greatly exceeds the limit value placed on an inorganic fluoride (OEL = 2.5 mg/kg bw/day F) and as such is not considered to be relevant for risk assessment.

Justification for selection of Effect on developmental toxicity: via oral route:
Study provides the key value for risk assessment.

Justification for classification or non-classification

No classification is proposed for reprotoxicity or developmental toxicity of disodium fluorophosphate. Some limited effects were noted in the studies however, these have been linked to maternal toxicity and therefore the substance is not suspected to be a reproductive or developmental toxicant.