Reaction mass of cerium phosphate and lanthanum phosphate and terbium phosphate

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:

from 26-NOV-2012 to 28-MAY-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: the study was performed according to OECD guideline and GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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:

cerium(3+) lanthanum(3+) terbium(3+) triphosphate

EC Number:

915-152-1

Molecular formula:

(La,Ce,Tb)PO4

IUPAC Name:

cerium(3+) lanthanum(3+) terbium(3+) triphosphate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: on the first day of treatment, the males were approximately 10 weeks old and the females were approximately 9 weeks old
- Weight at study initiation: on the first day of treatment, the males had a mean body weight of 387 g (range: 328 g to 436 g) and the females had a mean body weight of 222 g (range: 192 g to 249 g)
- Fasting period before study: no
- Housing: the females were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). The males were individually housed, except during mating, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray containing autoclaved sawdust (SICSA, Alfortville, France) was placed under each cage. Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 2537604 and 8788036 (SSNIFF Spezialdiäten GmbH, Soest, Germany), distributed weekly
- Water: free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%,
- Air changes: about 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: from 27 November 2012 (day of arrival of the animals) to 27 January 2013 (necropsy of last females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) methylcellulose aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The test item dose formulations were prepared on a daily basis and delivered to the study room at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): a stable suspension was obtained in this aqueous vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL, respectively for the dose levels of 100, 300 and 1000 mg/kg bw/day
- Amount of vehicle: a constant dosage-volume of 5 mL/kg/day was used
- Lot/batch no. (if required): the vehicle was prepared using: drinking water treated by reverse osmosis using ELIX 5 (Millipore SA) and methylcellulose (batch No. 079K0054)

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: each female was placed with the same male until mating occurred (and with a second male when the first male died during the mating period).
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 p.c.
- Further matings after two unsuccessful attempts: not concerned
- After successful mating each pregnant female was caged: individually housed
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of the test item in the dose formulations were quantified by ICP-MS, a validated analytical method. It consisted of sampling accurate volume of dose formulations, mineralized the sample using acid and micro wave, and diluting it appropriately with diluent to reach the nominal concentration of measurement.
Before the start of treatment, the suitability of the dose formulation process was confirmed. The homogeneity was determined on a range of dose formulations prepared at levels which covered the lowest and highest concentrations proposed for use in this study.
The concentration of the test item in samples of each control and test item dose formulation used in weeks 1, 3 and 6 was determined and were found within an acceptable range of variations (-11.3 % to -4.5 % when compared to nominal values (+/- 15%).

Duration of treatment / exposure:

- in the males: 2 weeks before mating, during the mating period, and until sacrifice (at least 5 weeks in total)
- in the females: 2 weeks before mating, during the mating period, during gestation, during lactation until day 5 p.p. inclusive

Frequency of treatment:

Once a day, at approximately the same time.

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 12 weeks old for males and approximately 11 weeks old for females.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal

No. of animals per sex per dose:

10 males and 10 females per dose-level group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: following the results of a previous 2-week toxicity study (CiToxLAB France/Study No. 39259 TSR) in which no obvious test item-related effects occurred during the study up to higher dose tested (1000 mg/kd bw/day).

Positive control:

not applicable (not required)

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period. Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.
The first five surviving males and females to be sacrificed on day 6 p.p. from each group were evaluated with a functional observation battery once at the end of the treatment period. This included a detailed clinical examination, the assessment of reactivity to manipulation and to different stimuli and motor activity. All animals were observed in the cage, in the hand and in the standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice. The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p..

FOOD CONSUMPTION
The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the mating period. The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of the mating period, during gestation for the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval days 1-5 p.p..
During the mating period, food consumption was not measured for males or females.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the females were mated.

Sperm parameters (parental animals):

Parameters examined in all male parental generation: testis weight, epididymis weight
Parameters examined in the first five sacrificed as scheduled males: stages of spermatogenesis, histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
weight gain, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, , physical or behavioural abnormalities.
The pups were observed daily for clinical signs, abnormal behavior and external abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes.
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups. For all pups found dead, special attention was paid to the reproductive organs and to whether the pup has fed (e.g. presence of milk in the stomach). No tissues were preserved.

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
- males: after the end of the mating period (at least 5 weeks of treatment in total),
- females: on day 6 p.p..

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all F0 animals including the male that died during the study. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females sacrificed as scheduled on day 6 p.p..

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
Surviving pups were sacrificed by an intraperitoneal injection of sodium pentobarbital on day 5 p.p..

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups sacrificed on day 5 p.p.. No tissues were preserved.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed

Statistics:

Statistical analyses were performed on body weight, food consumption, reproductive data, hematology, blood biochemistry and organ weights.

Reproductive indices:

Data are recorded and calculated using computerized validated software (Reprotox version B.1 and PathData).
Data are expressed as group mean values ± standard deviation (body weight, body weight change, food consumption, number of corpora lutea, number of implantation sites, number of pups and pup body weight, gestation length) or as proportions (pre implantation loss, post-implantation loss, pup observations, mating index, fertility index, gestation index, live birth index, viability index). Whenever necessary, the experimental unit of comparison was the litter.
The following parameters were calculated:
- pre-implantation loss,
- post-implantation loss (manually calculated),
- mating index,
- fertility index,
- gestation index.

Offspring viability indices:

The following parameters were calculated:
- live birth index,
- viability index on day 4 p.p.

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:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male given 1000 mg/kg/day was found dead on day 22. The death was considered to be related to the gavage procedure and thus not test item-related. No other animals died during the study.
There were no test item-related clinical signs in males and females from all groups.
There were no toxicological relevant effects on FOB and motor activity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no statistically significant effects of treatment with the test item on mean body weight and mean body weight gain in males and females from all groups.
There were no effects of treatment with the test item on mean food consumption in males and females from all groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test item-related effects on mean mating and fertility data.
The slightly higher mean pre-coital time noted at 1000 mg/kg/day was due to one female which was blocked in diestrous for several days and mated with the second male (as the first male died during the mating period) after 13 days. This was considered to be incidental.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No data on sperm but careful histology of testis was done and no effect due to treatment was observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no test item-related effects on mean mating and fertility data.
There were no toxicologically relevant effects of test item treatment on mean delivery data.
The slightly apparent higher mean percentage of pre-implantation loss noted at 1000 mg/kg/day was particularly due to two females:
- one female which lost five implants, vs. up to 4 in the control group,
- one female which lost three implants only but had a high percentage of pre-implantation loss (only eight corpora lutea ).
Globally these percentage are comparable to historical controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related organ weight differences.
When compared with controls, there were not statistically significant increases in mean absolute and relative-to-body spleen weights in test item-treated males. In the absence of microscopic correlates, these poorly dose-related differences were considered not to be related to test item treatment.
The other mean organ weight differences were considered not to be test item treatment-related in the absence of dose-relationships and microscopic correlates and because of the low magnitude in these differences.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no macroscopic test item-related findings in males and females from treated group as compared to controls animals.
The few macroscopic findings were considered as not test item effects because they were consistent with spontaneously occurring findings described in the literature or their appearance was similar to findings found in controls.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no microscopic test item-related findings in any males or females as compared to control animals.
The microscopic findings were considered not as related to the test item because they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No relevant effects up to highest dose tested

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)

VIABILITY (OFFSPRING)
There were no effects of treatment with the test item on pup viability by day 5 p.p..

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs observed due to treatment with the test item by day 5 p.p..

BODY WEIGHT (OFFSPRING)
There were no effects of treatment with test item on mean body weight and mean body weight change.

GROSS PATHOLOGY (OFFSPRING)
There were no test item-related macroscopic findings at pup necropsy (no dose-relationship and slight incidences).

OTHER FINDINGS (OFFSPRING)
There were no effects of treatment on the percentage of male pups at birth.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No relevant effects up to highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 2: Parental mean body weights and mean body weight changes (g)

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000		0	100	300	1000
Pre-mating
Day 1	383	387 (+1)	390 (+2)	389 (+2)		222	222 (0)	223 (0)	222 (0)
Day 15	437	446 (+2)	454 (+4)	456 (+4)		242	244 (+1)	246 (+2)	246 (+2)
Day 36	464	493 (+6)	494 (+6)	499 (+8)		/	/	/	/
Days 1 - 15	+54	+59	+64	+67		+20	+22	+24	+24
Days 15 - 36	+27	+47	+40	+38		/	/	/	/
Days 1 - 36	+81	+106 (+31)	+104 (+28)	+107 (+32)		/	/	/	/
Gestation
Day 0p.c.	/	/	/	/		252	250	248	256
Day 20p.c.	/	/	/	/		421	415	403	416
Days 0 - 20p.c.	/	/	/	/		+169	+165	+155	+160
Lactation
Day 1p.p.	/	/	/	/		316	313	305	321
Day 5p.p.	/	/	/	/		333	321	319	333
Days 1 - 5p.p.	/	/	/	/		+17	+7	+14	+12

p.c.: post-coitum, p.p.: post-partum; /: not applicable,

( ): in brackets percentage of changevs.controls.

Table 3: Mating and fertility data

Dose-level (mg/kg/day)	0	100	300	1000
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	9^a
Number of females mated	10	10	10	10
Mating index (%)	100	100	100	100
Mean number of days taken to mate (pre-coital time)	2.9	2.7	2.4	3.9
Number of pregnant females	10	10	10	10
Fertility index (%)	100	100	100	95^a
Number of females with live born pup	10	10	10	10
Gestation index (%)	100	100	100	100

a: male A25231 died during the mating period before mating.

Table 4 : delivery data

Dose-level (mg/kg/day)	0	100	300	1000
Number of females which delivered	10	10	10	10
Mean duration of gestation (days)	21.2	21.5	21.3	21.4
Mean number ofcorpora luteaper female	16.6	17.4	16.8	16.6
Mean number of implantationsper female	15.8	16.5	16.2	14.9
Mean pre-implantation lossper female(%) (number of affected females)	5.1 (4)	5.0 (4)	3.6 (3)	11.3 (5)
Mean number of pups deliveredper female	15.1	15.2	14.7	13.9
Mean post-implantation lossper female(%)^a	4.1	8.0	9.3	7.4

a: manually calculated, no statistics performed.

Table 5: Mean pup body weights and body weight changes (g)

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Body weight
. Day 1p.p.	7.0	7.3	6.9	7.0	6.6	6.7	6.4	6.9
. Day 5p.p.	11.0	11.7	11.0	11.6	10.6	11.1	10.5	11.4
Body weight change
Days 1-5p.p.	+4.0	+4.4	+4.1	+4.5	+4.0	+4.4	+4.1	+4.5

Table 6: pup sex ratios per group at birth

Dose-level (mg/kg/day)	0	100	300	1000
Percentage of males at birth (%)	49.7	58.6	55.1	50.4

Table 7: Macroscopic post-mortem examination of pups

Dose level (mg/kg/day)	0	100	300	1000
Dead pups
Number of pups (litter) examined	0	5 (5)	2 (2)	1 (1)
. Autolysis	0	2 (2)	2 (2)	0
. Stomach: absence of milk	0	2 (2)	0	1 (1)
. Abdominal cavity: fluid-filled abdomen	0	1 (1)	0	0
Total	0	4 (4)	2 (2)	1 (1)
Scheduled sacrifice pups
Number of pups (litter) examined	151 (10)	146 (10)	142 (10)	137 (10)
. Stomach: absence of milk	0	3 (3)	2 (2)	0
Total	0	3 (3)	2 (2)	0

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study, the test item did not affect the parent animals and the pups by day 5 p.p. after treatment up to 1000 mg/kg/day.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test performed according to OECD test guideline 422 and in compliance with GLP,(CiTOXLab, 2013), the potential general and reproductive or developmental toxicity of Reaction mass of Lanthanum Phosphate and Cerium Phosphate and Terbium Phosphate were tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0 (vehicle), 100, 300 or 1000 mg/kg/day in 0.5% methylcellulose aqueous solution. The study was scored at validity 1 according to Klimisch criteria.

No treatment-related death or clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain, food consumption, FOB and motor activity, hematology and blood biochemistry at any dose level. At histopathology, there were no test item-related mean organ weight differences, macroscopic or microscopic findings

There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.

Pups showed no effects of treatment on survival and the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio. There were no treatment-related macroscopic abnormalities.

 

Based on the experimental conditions of this study, the test item did not affect the parent animals and the pups by day 5 p.p. after treatment up to 1000 mg/kg/day.

Thus the NOAEL for parental systemic toxicity and for reproductive performance (mating and fertility) is 1000 mg/kg bw/day.

As no effect were observed in the pups by day 5 p.p., NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day.

No classification for repeat-dose toxicity or reproductive or developmental toxicity is warranted based on the absence of relevant effects in this study, according to the EU/UN GHS criteria.

This study is classified as acceptable. It satisfies the OECD 422 guideline requirements on repeated dose toxicity testing and reproduction/developmental toxicity screening.