Lanthanum fluoride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 October 2012 to 24 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lanthanum fluoride

EC Number:

237-252-8

EC Name:

Lanthanum fluoride

Cas Number:

13709-38-1

Molecular formula:

F3La

IUPAC Name:

lanthanum fluoride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appearance: white powder
- Storage conditions: ambient, protected from light

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-10 weeks (treatment initiation).
- Weight at study initiation: 301 - 369 g (males); 232 - 285 g (females) at treatment initiation.
- Housing: Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates. Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 22 °C
- Humidity (%): 32 - 83 %
- Air changes (per hr): 10 air changes per hour (minimum)
- Photoperiod (hrs dark / hrs light): a 12 hour light/dark cycle was maintained
- Environmental enrichment: Chewing objects and hiding devices were provided to the animals as appropriate for psychological/environmental enrichment.

IN-LIFE DATES: From 29 October 2012 to 17 December 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% (w/v) Carboxymethylcellulose (high viscosity) in Milli-Q water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. They were prepared by adding an appropriate amount of vehicle to the required amount of test material and were mixed by a high sheer mixer and magnetic stirring until a visibly homogenous formulation was obtained. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes prior to dosing and continuously during dosing. All formulations were used within the 8-day stability period that had been previously established.

- Dose volume: 10 mL per kg body weight.

- The volume administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.

Details on mating procedure:

- M/F ratio per cage: 1:1. A few days prior to the initiation of mating, the males were separated into individual grid bottom cages. Pairings were on a one male to one female basis.
- Length of cohabitation: Animals were paired in ascending numerical order within each group. Each female was transferred to the cage of its appropriate co-group male near the end of the work day, where it remained until mating had occurred. Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of sperm in such a lavage was designated Day 0 of gestation.
The time taken for each female to show a positive mating sign was evaluated.
- Proof of pregnancy: Presence of sperm in the vaginal lavage was designated Day 0 of gestation.
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From each formulation, duplicate sets of top, middle and bottom samples were taken at each sampling time point and sent to the analytical laboratory for analysis (duplicate middle samples only obtained from Control formulations). Additional triplicate samples from the top, middle and bottom (middle sample only from control) were also taken as back-up samples. The results of the sample concentration analyses were considered acceptable if all results were within ± 15 % of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤ 10 % for each group.

- Preparation of test solutions: the appropriate amount of formulation was weighed into a digestion vessel and digested in aproximately 10 mL of 69 % nitric acid. Once digested, the samples were transferred to a 50 mL volumetric flask with washings made to volume in 2 % nitric acid. The samples were further diluted in 2 % nitric acid to give sample solution concentrations within the range of the calibration standard used. Samples and standards were analysed by ICP-OES.

- Preparation of calibration standards: 20 mg of test material was added to a digestion vessel and digested in approximately 10 mL of 69 % nitric acid. Once digested the solution was transferred to a 100 mL volumetric flask with washings and made to volume in 2 % nitric acid to obtain a stock solution with a concentration of 0.200 mg/mL lanthanum fluoride. The stock solution was diluted volumetrically in 2 % nitric acid to obtain target concentrations of approximately 0.00100, and 0.0100 mg/mL lanthanum fluoride.

- ICP-OES conditions:
> Instrument: Perkin Elmer Optima 8000 ICP-OES
> Elements and wavelength: La (398.852 nm)
> No. of replicates: 3
> Delay time: 60 seconds
> Read time: set to auto
> Minimum read time: 1 second
> Maximum read time: 5 seconds
> Points per peak: 7
> Plasma conditions:
Plasma: 8 L/min
Auxiliary: 0.2 L/min
Nebuliser: 0.7 L/min
RF power: 1300 Watts
Plasma viewing mode: radial
> Peristatic pump sample flow rate: 1.50 mL/min
> Wash:
Frequency: between samples
Flow rate: 1.50 mL/min
Normal time: 30 seconds
Wash solvent: Milli-Q water
> Diluent: 69 % nitric acid (for digestion); 2 % nitric acid (for dilutions)
> Microwave digestion:
Ramp: 20 min to 200 °C
Hold: 20 min at 200 °C
Cool down: 45 min

- Calculations: a calibration curve was constructed by plotting the mean peak areas of the standards (including the calibration blank) against the relevant lanthanum fluoride concentration in mg/mL. The concentration of lanthanum fluoride in the test samples was determined by interpolation of this line.

Concentration of lanthanum fluoride in formulations (mg/mL) = (C x D x P) / W

Where
C = concentration of the sample solution obtained by interpolation from the calibration line (mg/mL)
D = dilution factor
P = density of the formulation (g/mL)
W = weight of aliquot (g)

- The limit of detection (LOD) estimated from an analyte response at the level of 3.3 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 1.59 x10^-6 mg/mL Lanthanum Fluoride.
- The limit of quantification (LOQ) estimated from the an analyte response at the level of 10 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 4.81 x10^-6 mg/mL Lanthanum Fluoride.

Duration of treatment / exposure:

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment).
* Animals 41 (Group 1 female) and 59 (Group 2 female) were not dosed on their respective days of parturition due to the animals starting parturition prior to the commencement of dosing that day.

Frequency of treatment:

Animals were dosed once daily.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected after a review of existing relevant toxicological data. Including a separate 14-day dose range finding study, where dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
- Animal assignment: On arrival from the suppliers, the animals were housed in cages. The cages were suspended on a series of racks. Male and female cages were racked separately. Cages were allocated to treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups. During pre-trial Animals 45 (Group 1 female) and 77 (Group 4 female) were replaced with spare Animals 83 and 84, respectively, due to findings in the pre-trial ophthalmic examinations. Animals 45 and 77 were not used and were therefore considered not to be part of this study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for viability early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded one week prior to the start of treatment. From the start of treatment, the individual body weights were recorded daily.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating.
After pairing, the female food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-4 of lactation. Male food consumption did not recommence after pairing for mating.

Litter observations:

The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.
Where practicable, any pups that were found dead or killed during lactation were sexed and appropriately examined as above. All pups were externally normal and were discarded following examination.

Postmortem examinations (parental animals):

The males were killed when mating was completed and the animals had been dosed for at least 4 weeks (Study Day 30).
The females and litters were killed between Day 5 and 7 of lactation (Study Days 43, 46 - 47 and 50).
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

GROSS PATHOLOGY: Yes
All adult animals surviving to scheduled euthanasia were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded and the total numbers of corpora lutea graviditatis were counted.

ORGAN WEIGHTS: Yes
The following organs were weighed at necropsy for all adult animals surviving to terminal kill: Brain, Epididymides, Adrenal glands, Pituitary gland, Prostate gland, Thyroid glands, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Uterus.

HISTOPATHOLOGY: Yes
Representative samples of the following tissues were collected from all adult animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated:
Aorta, Blood smear* (Animals sacrificed prematurely only), Bone marrow smear# , Bone marrow (femur, sternum), Femur, Rib, Sternum, Brain, Cervix, Epididymides, Eyes~, Adrenals, Harderian glands~ , Lacrimal glands, Mammary gland, Parathyroid glands, Pituitary gland, Prostate gland, Salivary glands, Seminal vesicle, Thyroids, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidneys, Large intestine (caecum, colon, rectum), Larynx, Liver, Lung, Mandibular lymph node, Mesenteric lymph node, Skeletal muscle, Nasal cavity, Optic nerve~, Sciatic nerve, Oesophagus, Ovaries, Oviducts, Pancreas, Pharynx, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Testes+, Thymus, Tongue, Trachea, Ureter x2, Urinary bladder, Uterus, Vagina.
* Air dried
# Fixed in methanol and then air dried
~ Preserved in Davidson’s fixative.
+ Preserved in Modified Davidson’s fixative.

- Bone Marrow Smears
Duplicate bone marrow smears were taken at necropsy from all adult animals. Both bone marrow smears were stained using May-Grunwald-Giemsa as the Romanowsky stain. Bone marrow smears were not evaluated.

- Histopathology
The tissues, as listed above (except animal identification, nasal cavity, blood smears and bone marrow smears), were processed to paraffin wax block from selected animals. The testes of all male animals were processed to wax impregnation. Sections were cut 4-6 µm thick, stained with haematoxylin and eosin from 5 males and 5 females in the Control and High dose groups (the same animals that were used for laboratory investigations). An additional PAS-Haematoxylin stained slide was prepared from the testes and epididymides of the males.

Postmortem examinations (offspring):

Where practicable, animals found dead or killed prematurely were sexed, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Surviving pups were also examined for externally visible abnormalities. All pups were then discarded.

Statistics:

Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Selected body weight and food consumption data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Reproductive indices:

Fertility index (male and female), gestation index, birth index and live birth index were calculated.

The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which the first pups were born) was designated as Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest which was replaced when it became soiled.

Offspring viability indices:

Viability index was calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
There were no premature decedents on this study. Treatment at levels up to 1000 mg/kg/day in males and females produced no clinical observations that were considered related to treatment with the test material. In both sexes and in all dose groups there was high incidence of soft faeces observed. As this was present in almost all Control animals, this was considered to be an effect caused by the vehicle and not related to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
In the treated males the group mean body weight gains appeared to be slightly reduced when compared to Controls. The high standard deviation in Controls shows a high degree of variation between the individual animals within this group. The group mean gain has also been skewed by 2 outlier males who had very high body weight gains throughout the duration of the study (Animals 2 and 10), when compared to the other males in this group. Therefore this apparent difference in body weight gain is considered incidental and not related to treatment with the test material. At levels up to 1000 mg/kg/day the group mean body weight gain for females prior to mating and throughout gestation and lactation were similar to Control.

FOOD CONSUMPTION
Group mean food consumption prior to pairing for mating was similar to Controls in both males and females. The group mean food consumption in all treated females was similar to Controls during gestation and lactation.

MATING PERFORMANCE, FERTILITY AND DURATION OF GESTATION AND LITTER SIZE
In all treated groups the mating performance, fertility indices and duration of gestation were similar to Controls. In all treated groups the mean number of corpora lutea and the mean number of implantation sites were similar to Controls. In all treated groups the mean number of pups born per litter and the mean number of pups per litter on Days 0, 1 and 4 of lactation was similar to Controls.

OBSERVATIONS AMONG DAMS
The type and distribution of observations amongst dams during lactation did not indicate any association with treatment.

ORGAN WEIGHTS
In all treated animals the organ weights were similar to Controls. Any slight intergroup differences in the organ weights were too small to be related to administration of the test material.

GROSS PATHOLOGY
No test material-related gross findings were noted. The gross findings observed were considered incidental and of the nature commonly observed in this strain and age of rat.

HISTOPATHOLOGY
There were no treatment-related microscopic findings. All of the histopathological findings encountered were considered to have arisen spontaneously or at post mortem. The incidence and severity of microscopic findings were similar in control and treated animals and, therefore, were considered unrelated to administration of the test material.

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

(reproductive)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

NOAEL

Remarks:

(toxicicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

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:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

LITTER SURVIVAL, LITTER AND PUP WEIGHTS
In all treated groups the birth index, live birth index and viability index was similar to Controls. There was a higher than expected incidence of dams that had a total litter loss: 2/10 in Control, 2/10 at 100 mg/kg/day, 3/10 at 300 mg/kg/day and none at 1000 mg/kg/day. When the groups are combined it can be seen that 7/40 (17.5 %) of females had a total litter loss. This is within the Historical Control Range for this species and strain where up to 20 % of dams have incurred a total litter loss in one study. The high incidence of litter loss in Controls along with the absence of litter losses at 1000 mg/kg/day indicates that this is not related to treatment with the test material. With the exception of the dams that lost their entire litter, the group mean litter size remained comparable to Controls throughout lactation. In all treated groups the group mean litter weight and the mean of litter mean pup weight were similar to Controls throughout lactation.

OBSERVATIONS AMONG PUPS
The type and distribution of observations amongst litters during lactation did not indicate any association with treatment.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 2: Mating Performance and Fertility Indices

No of nights to positive mating	Group / Dose level (mg/kg/day)
	1 (0)	2 (100)	3 (300)	4 (1000)
1	Number of animals (not becoming pregnant)
	2	4	2	3
2	4	1	2	2
3	0	2	1	2
4	4	3	4	3
7	0	0	1	0
No clear indication of mating	0	0	0	0
Median no. of nights to positive mating	2	2.5	3.5	2.5
No. passing one oestrous	0	0	1	1
No. of males paired	10	10	10	10
No. of siring males	10	10	10	10
Male fertility index (%)	100	100	100	100
No. of females paired	10	10	10	10
No. pregnant	10	10	10	10
Female fertility index (%)	100	100	100	100

 

Table 3: Group Mean Duration of Gestation and Overall Litter Performance

	Group / Dose level (mg/kg/day)
	1 (0)	2 (100)	3 (300)	4 (1000)
No pregnant	10	10	10	10
Duration of gestation (days)
21	3	5	4	6
22	7	5	5	4
23	0	0	1	0
Mean duration	21.7	21.5	21.7	21.4
No. of females producing a live litter	10	10	10	10
Gestation index (%)	100	100	100	100
Mean no. of implant sites* per pregnancy	16.0	16.4	17.3	16.7
Mean no. of corpora lutea sites* per pregnancy	20.3	17.9	18.7	19.9
Mean total no. of pups born* per litter	15.0	14.9	16.0	15.3
Mean no. of live pups* per litter on
Day 0 of lactation	14.5	14.9	16.0	15.2
Day 1 of lactation	12.8	14.8	15.6	14.7
Day 4 of lactation	12.6	14.0	15.0	13.6
Total no. of males* on day 1 of lactation (%)	44 (43)	65 (55)	64 (59)	64 (44)
Total no. of females* on day 1 of lactation (%)	58 (57)	53 (45)	45 (41)	83 (56)

* excludes liters where all pups died

Table 4: Group Mean F1 Survival Indices

		Group / Dose level (mg/kg/day)
		1 (0)	2 (100)	3 (300)	4 (1000)
Birth index	Mean litter index (%)	89	92	92	91
	No. losing > 2 pups	3	2	3	2
	No. of litters	10	10	10	10
Live birth index	Mean litter index (%)	96	100	99	99
	No. losing > 1 pup	2	0	1	0
	No. of litters	10	10	10	10
Viability index (days 0-4)	Mean litter index (%)	72	76	66	90
	No. losing > 3 pups	3	3	3	2
	No. of litters	10	10	10	10

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the reproductive No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day, for both males and
females.

Executive summary:

The toxicity of the test material to reproduction was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 422. During the study groups of 10 male and 10 female rats were administered test material, by oral gavage, at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment).

The following reproductive and viability indices were evaluated; fertility index (male and female), gestation index, birth index, live birth index and viability. In addition, the following parameters and end points were evaluated: clinical signs, body weights, body weight changes, food consumption, gross necropsy findings, organ weights, histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

At dose levels up to 1000 mg/kg/day, there were no effects of treatment on clinical signs, body weights, body weight changes, food consumption, gross necropsy findings, organ weights, histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Therefore, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day, for both males and females.