Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-10-2012 - 05-04-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 422.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 11 weeks
- Weight at study initiation: males: 337 to 379 g (mean 361 g); females: 185 to 221 g (mean 198 g)
- Fasting period before study: no data
- Housing: In groups of five in Markolon type-4 cages during the first days of acclimatization and then individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light/ 12 hour dark cycle
Route of administration:
oral: gavage
Vehicle:
other: bidistilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly.

Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories Study D57908), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

Lanthanum acetate was weighed into a glass beaker on a tared precision balance, vehicle was added (w/v) initially stirred with a magnetic stirrer before adding the remaining vehicle and stirring for approximately 30 minutes until homogeneously suspended. Daily aliquots were portioned into glass containers. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Storage of dose formulations:
Dose formulations were stored at room temperature (20±5 °C) in glass beakers, away from direct sunlight.

Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study D57910, dose formulations were stable for at least four weeks when stored in these conditions.
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females will be removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed

The day of mating was designated day 0 post-coitum.

If a female did not mate during the 14-day pairing period, this female was paired with a male of the same group which had already mated successfully. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed, and if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams will be allowed to give birth and rear their kittens (F1 pups) up to day 5 post-partum. Day 0 was designated as the day on which a female had delivered all her pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stock solutions of lanthanum acetate in purified water were prepared for external standard calibration. Then, the mixture was sonicated for at least 5 minutes and the flask was brought to volume with purified water to yield a solution with a concentration of 452.0 µg/mL (a correction factor of 1.08 was taken into account). Aliquots of this stock standard solution were used to prepare working standard solutions in purified water with a concentration range of 11.30 to 90.41 µg/mL.

Analysis of samples:
The samples received were dissolved in purified water by sonication for at least 5 minutes and then diluted to volume with purified water. Where necessary, sample solutions were further diluted with purified water into the calibration range.

Method of analytical verification: High performance liquid chromatographic determination

Evaluation of results:
Injected samples were quantified by comparing peak areas of lanthanum acetate with reference to the calibration curve. The latter was obtained by correlation of the peak areas of the working standards with their corresponding concentrations [µg/mL], using the linear regression model following equation:
y = a + (b * x)
y = response of lanthanum acetate
a = intercept derived from linear regression of calibration data
b = slope derived from linear regression of calibration data
x = actual concentration of lanthanum acetate in sample aliquot [µg/mL]

Results and conclusion:
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ± 20% variation from the calibration curve derived by linear regression analysis. The regression coefficients (R²) calculated were found to be better than 0.99.

The lanthanum acetate peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of lanthanum acetate and, therefore, the absence of the test item in the vehicle control samples (bidistilled water) was confirmed.

The application formulations investigated during the study were found to comprise lanthanum acetate in the range of 88.6% to 102.6% and, thus, the required content limit of ± 20% with reference to the nominal content was met. The homogeneous distribution of lanthanum acetate in the preparations was approved because single results found did not deviate more than 4.4% (acceptance criterion: < 15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item lanthanum acetate and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
Males: 46 days
Females: approximately 7 weeks (49 days)
Frequency of treatment:
Once daily
Details on study schedule:
- First test item administration:
Males and females: day 1 of pre-pairing
- Pre-pairing:
Males and females: 14 days
-Pairing::
Males and females: 14 days maximum
-Gestation:
Females: approximately 21 days
- Treatment ends:
Males: on day before sacrifice
Females: on day 4 post partum
Remarks:
Doses / Concentrations:
0, 110, 330, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
12 (plus 2 extra per sex in a reserve group)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study D57908, which showed reduced food consumption and body weight development at 1000 mg/kg/day and a trend for lower body weights at 300 mg/kg/day.

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, during acclimatization and up to day of necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, during acclimatization and up to day of necropsy. Additionally females will be observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
- Animals were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual repiratory pattern) as well as changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypic or bizarre behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: From treatment start to day before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Males: weekly during pre-pairing and after pairing periods
Females: pre-pairing period days 1-8 and 8-13; gestation days 0-7, 7-14 and 14-21 post coitum, and lactation days 1-4
No food consumption was recorded during the pairing period.

HAEMATOLOGY: Yes
- Blood samples were obtained on day 14 of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Parameters checked were: complete blood cell count (erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, leukocyte count total, differential leukocyte count, platelet count) and coagulation (prothrombin time and activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Parameters checked were: glucose, urea, creatinine, bilirubin total, cholesterol total, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorus, protein total, albumin, globulin, albumin/globulin ratio

NEUROBEHAVIOURAL EXAMINATION: Yes
At one time during the study (males in the fourth week of treatment and females on day 3 or 4 post partum), relevant parameters from a modified Irwin screen test were performed with five P generation males and five P generation females from each group in place of the usual weekly behavioral observation. This assessment was conducted following the daily dose administration. Any abnormal findings were recorded and, where appropriate, graded in severity.

Grip strength:
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge. The animals were placed with the forepaws inside a triangular rasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

Locomotor activity:
Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH and DeMeTec GmbH Activity Monitor System. Animals were monitored for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

OTHER: Organ weights:
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

In addition, from 5 males and 5 females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate and their wet weight was taken: adrenal glands (weighed as pairs), brain, heart, kidneys (weighed as pairs), liver, thymus, spleen
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight
Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post-partum.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Dams and pups were sacrificed on day 5 post-partum.
At the scheduled sacrifice, terminal body weights were measured and all animals were sacrificed by an injection of sodium pentoarbital. All P generation animals were exsanguinated.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All parent animals and pups were examined macroscopically for any structural changes.For the parent animals, special attention was directed at the organs of the reproductive system.The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites

HISTOPATHOLOGY:
Histotechnique:
All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

Histopathology:
The tissue from all parental males was preserved in neutral phosphate buffered 4% formaldehyde solution: prostate, seminal vesicles with coagulating glan, testes and epididymides.
Ovaries from parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.
From all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
gross lesions, brain, spinal cord, small and large intestines, stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroids and parathyroids if possible, trachea and lungs, uterus with vagina, urinary bladder, lymph nodes, peripheral nerve, bone marrow
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Because of possible test item-related findings noted in the high dose group, the stomach of both sexes were examined from five animals in the intermediate groups, as well as a small number of other organs from animals which did not mate or did not litter.
A histopathology peer review was performed. The assessment of the study pathologist and reviewing pathologist compared favorably.
Postmortem examinations (offspring):
The litters were examined for any gross anomalies.
Statistics:
Following statistical methods were used to analyze food consumption, body weights and reproduction data:
- means and standard deviations of various data were calculated
- the Dunnett-test (many to one t-test) based on a pooled variance estimate if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex
- The Steel-test (many-one rank test) were applied instead of the Dunnett-test when the data were not assumed to follow a normal distribution
- Fischer's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios
Offspring viability indices:
From the on-line recorded reproduction data, the following parameters were calculated: viability indices.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
All males and females survived until their respective scheduled necropsies.

Clinical observations:
Males:
There were no test-item related findings in any male rat during the pre-pairing, pairing or after pairing periods.
During the pre-pairing period, one male treated with 1000 mg/kg/day had a transient slight decrease of activity. There were no findings in other males during the pre-pairing period.

During the pre-pairing period, small areas of localized hair loss were recorded in a few males treated with 1000 mg/kg/day. Breathing noises were recorded intermittently in one or two males at this dose level, as was salivation. Transient breathing noise was also recorded in one male treated with 330 mg/kg/day. These findings were not considered to be related to the treatment with the test item. Males treated with 110 mg/kg/day were unaffected.

During the after pairing period, slight hair loss was noted in one male treated with 1000 mg/kg/day and transient breathing noise was noted in one male treated with 330 mg/kg/day. The remaining males were without findings.

Females:
There were no test item-related findings in any female rat during the pre-pairing, pairing, gestation or lactation periods.

There were no symptoms noted in any female during the pre-pairing and pairing periods. Breathing noise was noted on one occasion during the gestation period in one female treated with 1000 mg/kg/day and ruffled fur was noted on one occasion in one female treated with 110 mg/kg/day.

No symptoms were noted during the lactation period at any dose level.

BODY WEIGHT AND WEIGHT GAIN
Males (pre-pairing, pairing and after pairing periods):
A test item-related reduction in mean body weights was noted in males treated with 1000 mg/kg/day during the pre-pairing, pairing and after pairing periods. The mean body weight gain of these males was clearly lower during the pre-pairing period, but improved slightly during the pairing period and was considered to be unaffected during the after pairing period.

During the pre-pairing period, a trend for lower mean body weights were noted from day 3 onwards in males treated with 1000 mg/kg/day and attained statistical significance (p<0.05) from days 7 to 13. The mean body weight gain of these males during this period was significantly lower from days 2 to 14 (p<0.01).

The mean body weight of these males was significantly lower (p<0.05) during days 1 - 21 and day 24 of the pairing period. The body weights of these males remained lower than those of the controls males during the after pairing period and attained statistical significance (<0.05) on day 1, 2 and 4 of this period. The body weight gain of these males improved slightly during the pairing period and remained stable during the after pairing period and considered to be generally within range of normal variation.

Although the differences were not statistically significant, a trend for marginally lower body weights and mean body weight gain were noted in males treated with 330 mg/kg/day during the latter half of the pairing period and the after pairing period.

The mean body weights and mean body weight gain of the males treated with 110 mg/kg/day were generally similar to those of the control males.

Females (pre-pairing, pairing, gestation and lactation periods):
Lower mean body weights of female rats treated with 1000 mg/kg/day were noted on days 11 and 13 of the pre-pairing period (both p<0.05), and on day 5 of the gestation period (p<0.05). The mean body weight gain of these females was significantly lower from days 3-14 of pre-pairing (generally p<0.01), but improved during the gestation and lactation periods to levels similar to the control females.

The mean body weights and mean body weight gain of the females treated with 110 or 330 mg/kg/day compared favorably with those of the control females. Occasional incidences of statistical significance were considered to be of no toxicological relevance.

FOOD CONSUMPTION:
Males:
pre-pairing period: A statistically significant reduction in mean daily food consumption was noted during days 1 - 8 of the pre-pairing period in males treated with 1000 mg/kg/day (p<0.01). All other treated males were unaffected during this time interval and all males of all groups compared favorably during the second measurement interval (days 8 - 14).
after pairing period: There were no differences in mean daily food consumption during the after pairing period.

Females:
pre-pairing, gestation and lactation periods:
The mean daily food consumption of the females treated with 1000 mg/kg/day was significantly reduced during days 1 - 8 (p<0.01) and days 8 - 14 (p<0.05) of the pre-pairing period. A significant reduction in the females treated with 110 mg/kg/day during days 1 - 8 (p<0.05) of the pre-pairing period was not seen in the females treated with 330 mg/kg/day and therefore considered to be incidental.

With the exception of a slight reduction of food consumption during days 7 - 14 of the gestation period, the mean daily food consumption of the females treated with 1000 mg/kg/day was similar to that of the control females. The mean daily food consumption of the remaining dose groups was unaffected during the gestation period.

During the lactation period, there were no toxicologically relevant differences to the control values at any dose level.

HAEMATOLOGY
Males:
At 330 and 1000 mg/kg/day, slightly reduced levels of hemoglobin were statistically significant (p<0.05 and p<0.01, respectively) but remained within the range of the historical control values. The mean hematocrit was significantly lower in these groups but also remained within the ranges of the historical control data (p<0.05). A slight increase in the mean relative eosinophil count attained statistical significance (p<0.05) at 1000 mg/kg/day but remained with the historical control ranges. The mean thromboplastin time of the males treated with 330 mg/kg/day was very slightly elevated (p<0.05) when compared with the controls but in the absence of a dose-response relationship was considered to be incidental. These differences were considered to be without toxicological relevance.
The platelet count was significantly elevated in males treated with 1000 mg/kg/day (p<0.01) when compared with the controls, but remained with the range of historical control data and considered to be incidental.

At 110 mg/kg/day, the hematology parameters were similar to the control males.

Females:
At 1000 mg/kg/day, slightly reduced levels of hemoglobin and mean hematocrit were statistically significant (both p<0.01) but remained within the range of the historical control values. These differences were considered to be without toxicological relevance.

The mean platelet count was significantly elevated in females treated with 1000 mg/kg/day (p<0.05) when compared with the controls but remained within the range of the historical control data.

At 110 and 330 mg/kg/day, the hematology parameters were similar to the control females.

CLINICAL CHEMISTRY
Males:
At 330 and 1000 mg/kg/day, significantly reduced total bilirubin was noted in females (p<0.05 and p<0.01, respectively). Both values exceeded the lower limit of the historical control values. In view of the large variation in the individual values, a clear relationship with the test item treatment could not be conclusively drawn.

Significantly lower mean total protein (p<0.01), comprised of reduced albumin, p<0.05 and reduced globulin, p<0.01) were noted in the females treated with 1000 mg/kg/day. These latter changes were considered to be related to the treatment with the test item.

At 330 mg/kg/day, reduced total protein (p<0.01), comprised of reduced albumin (not significant) and reduced globulin (p<0.01) were noted and considered to test item related.

At 110 mg/kg/day, the clinical biochemistry parameters were similar to the control males.

NEUROBEHAVIOUR
There were no findings evident for males or females.

Grip strength:
The mean fore and hind limb strength values were unaffected at all dose levels.

The statistically significant increase noted in the hind limb grip strength of females treated with 110 mg/kg/day (p<0.05) was unrelated to dose and therefore considered to be incidental.

Locomotor activity:
The mean locomotor activity of males and females, recorded over 60 minutes in 10-minute intervals, compared favorably with that of the respective control values.

ORGAN WEIGHTS
In males and females, the mean absolute and relative organ weights were unaffected by the treatment with the test item.
A marginal reduction of thymus weights were noted in the males treated with 1000 mg/kg/day, but these were not associated with microscopical changes and therefore considered to be incidental. Females were unaffected.

GROSS PATHOLOGY
Males:
Red foci on the stomach were noted in two males treated with 330 mg/kg/day and three males treated with 1000 mg/kg/day, and were considered to be test item-related findings
Females:
There were no gross lesions that could be attributed to treatment with the test item. All findings were recorded to be within the range of normal background alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically, the following treatment related findings were recorded:
Stomach:
- Increase in incidence and severity of inflammatory cell infiltration in the submucosa to base of lamina propria consisting of eosinophils and/or lymphocytes was recorded in animals treated with 330 and 1000 mg/kg/day. The severity was slightly higher in males than females.
- Increase in incidence and severity of eosinophilic globule leukocyte in mucosa was recorded in animals treated with 330 or 1000 mg/kg/day.
- Atrophy of fundic gland mainly chief cells and/or parietal cells was recorded at minimal severity in males treated with 330 or 1000 mg/kg/day.
- Eosinophilic chief cells were recorded at minimal to slight severity in animals treated with 1000 mg/kg/day and males treated with 330 mg/kg/day.
- Increase in incidence and severity of epithelial vacuolation of squamous limiting ridge was recorded in males treated with 1000 mg/kg/day.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm staging:
The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. Results: No differences on the completeness of stages or cell populations of the testes were recorded between control and high dose animals. Sperm staging was not required for any male treated with 330 mg/kg/day since there were no females that failed to conceive or to litter, and ergo no indication of possible infertility.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility:
The mean precoital time of the females treated with 1000 mg/kg/day was slightly longer than that of the other treated females and the control females. One control male failed to mate (male number 5 which was initially paired with female no. 53) and was replaced by a second male that had mated successfully (male no. 3 which mated with female no. 53). Although no evidence of mating (in the form of a sperm plug or positive sperm smear) was for the control group and treated groups administered detected, female no. 53 littered successfully.

At 110 mg/kg/day, there were three females which did not litter: female no. 61 mated with male no. 13 but did not conceive, and female no. 71 mated with male no. 23 but did not conceive, and female no. 72 mated with male no. 24 but did not litter despite showing implantation sites.

At 330 mg/kg/day, all males and females paired were mated, all females conceived and all females littered.

At 1000 mg/kg/day, although there were two males that did not mate (male number 40 which was initially paired with female no. 88 and male no. 48 which was initially paired with female no. 96), both were replaced by other males that mated successfully and where the re-allocated dam littered successfully (male no. 38 which mated with female no. 88 and male no. 43 which mated with female no. 96). Evidence of mating (in the form of a sperm plug or positive sperm smear) was not seen in either of the females that failed to mate with males no. 40 or no. 48 or in the females of the replacement for the males (no. 38 or no. 43) that mated successfully (as evidenced by successful littering by the reallocated females). However, all females were eventually mated, all females conceived and all females littered.

In the absence of any relationship to dose, these findings were considered to be incidental. No effects on fertility (% mating, fertility index and conception rate) were observed at any dose level.

Duration of gestation: The duration of gestation was similar at all dose levels. (21.6, 21.4, 21.5 and 21.5 days for the control group and treated groups administered with 110 mg/kg/day, 330 mg/kg/day and 1000 mg/kg/day, respectively).

Corpora lutea count: The mean number of corpora lutea observed at all dose levels compared favorably with that of the controls (13.9, 12.8, 13.8 and 12.8 for the control group and treated groups administered with 110 mg/kg/day,

Implantation rate and post-implantation loss
The mean implantation rates of the test item-related and control groups compared favorably (13.3, 10.9, 12.0 and 12.0 for the control group and treated groups administered with 110 mg/kg/day, 330 mg/kg/day and 1000 mg/kg/day, respectively).

Although the mean post-implantation loss of the females treated with 1000 mg/kg/day was similar to the control females, the number of lost implantations as a percent of the total number was slightly higher in the females treated with 1000 mg/kg/day (7.5%, 7.1%, 5.6% and 11.1% for the control group and treated groups administered with 110 mg/kg/day, 330 mg/kg/day and 1000 mg/kg/day, respectively).

Litter size at first litter check:
When compared with the control females, the mean litter size of dams treated with 1000 mg/kg/day was marginally lower than that of the control females (12.3, 10.1, 11.4 and 10.7 living pups at first litter check for the control group and treated groups administered with 110 mg/kg/day, 330 mg/kg/day and 1000 mg/kg/day, respectively). This difference, in the absence of a clear dose-response relationship, was considered to be unrelated to the treatment.

Postnatal loss days 0 - 4 post-partum
There were no post-natal losses in dams treated with 330 mg/kg/day or 1000 mg/kg/day. A marginally elevated post-natal loss on the dams treated with 110 mg/kg/day was therefore considered to be incidental (0.2, 0.3, 0.0 and 0.0 for the control group and treated groups administered with 110 mg/kg/day, 330 mg/kg/day and 1000 mg/kg/day, respectively).

- Other findings:
No test-item related histological findings were recorded in the ovary of females which did not conceive (animal nos. 61 and 71, treated with 110 mg/kg/day) or the reproductive organs of their respective males (animal nos. 13 and 23, treated with 110 mg/kg/day) that were considered likely to be infertile. Female no. 72, treated with 110 mg/kg/day, mated successfully with male no. 24 but did not litter. The uterus was found to contain fetuses. The remainders of finding recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOAEL
Remarks:
(reproduction toxicity)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on a lack of toxicologically relevant effects on reproductive organs/tissues or reproductive performance of parental animals.
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
VIABILITY/CLINICAL SIGNS (OFFSPRING)
There were no abnormal external findings seen in any pup at the first litter check or during lactation.
Blue discoloration/hematoma of the genital region was noted in one pup at 110 mg/kg/day and one pup had no milk in the stomach. These findings were noted in the same litter. The low incidence and type of these findings suggested incidental occurrence.

There were no findings in pups of dams treated with 330 or 1000 mg/kg/day.

BODY WEIGHT (OFFSPRING)
The mean body weights of the pups on day 4 post-partum were considered to be unaffected by the treatment with the test item.

SEX RATIOS (OFFSPRING):
The sex ratios at first litter check were considered to be unaffected by the treatment with the test item.

GROSS PATHOLOGY (OFFSPRING)
There were no test item-related macroscopical findings in any pup necropsied on day 4 post-partum at any dose level.

Dose descriptor:
NOAEL
Remarks:
(developmental effects)
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on a lack of developmental effects on pups in any dose group.
Reproductive effects observed:
not specified

Results of dose range-finding study:

- Mortality/Viability:

All animals survived until scheduled necropsy.

- Clinical signs:

There were no clinical signs of toxicity at any dose level. Transient breathing noises were noted in one male at 1939.27 mg/kg bw/day. The remaining males and all females were without clinical signs.

- Food consumption:

There was a test item-related reduction in the mean daily food consumption in rats treated at 1939.27 mg/kg bw/day. This finding was more clearly exhibited in the females than in the males.

In males treated with 1939.27 mg/kg bw/day, the mean daily food consumption was -22.8% during days 1 -4 when compared with the control males. This improved to +2.3% and +20.9% during days 4 -7 and 7 -14, respectively. The females at this dose level consumed less feed than the control females: -21.0%, -14.4% and -9.8% during the measurement intervals of days 1 -4, 4 -7 and 7 -14, respectively.

The food consumption of males and females treated with 193.91 mg/kg bw/day or 581.72 mg/kg bw/day were generally similar to those of the controls.

- Body weights:

At 1939.27 mg/kg bw/day, clearly reduced mean body weights were noted in males and females on days 4, 7 and 14 of treatment.

The mean body weight gain was marginally lower in males treated with 581.72 mg/kg bw/day at the end of the treatment period when compared with the control males. Females at this dose level had marginally reduced mean body weight gain on days 7 and 14 of treatment.

At 193.91 mg/kg bw/day, the mean body weight gain was generally similar to that of the controls.

- Organ weights:

In males treated with 1939.27 mg/kg bw/day, slightly higher mean absolute adrenal weights were noted when compared with the control males. The mean absolute spleen weights of these rats were slightly elevated as well.

At 581.72 mg/kg bw/day, slightly higher mean absolute adrenal weights were noted.

The organ weights of females at all dose levels were similar to those of the controls.

- Macroscopic findings:

There were no test-item related macroscopical findings at any dose level.

At 1939.27 mg/kg bw/day, unilateral renal pelvis dilation was noted in one male and reddish foci on the thymus of one female.

At 581.72 mg/kg bw/day, unilateral renal pelvis dilation was noted in one female when compared with controls.

At 193.91 mag/kg bw/day, reddish discoloration of the thymus was seen in one male.

Based on the results of this 14 -day dose range-finding study, dose levels of 110, 330 or 1000 mg/kg body weight/day are proposed for the subsequent combined repeated dose toxicity study (with the reproduction/developmental toxicity screening test in the Han Wistar Rat) with lanthanum acetate.

Conclusions:
The NOAEL for reproduction/developmental toxicity was considered to be 1000 mg/kg/day (expressed ad lanthanum acetate anhydrous).
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction: oral

Braun (2013) performed a combined repeated dose toxicity study with reproduction/developmental toxicity screening test in RccHan: WIST (SPF) rats. This study is considered as key study and is scored as K1. Lanthanum acetate was administered to male rats for 46 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post-partum (approximately 49 days).

Animals were dosed at 110, 330 and 1000 mg/kg bw/day (expressed as anhydrous active ingredient).

Control animals were dosed with the vehicle (bi-distilled water) alone. There were no effects upon mortality of parental animals, no clinical findings (daily or weekly), no differences in the functional observational battery (including grip strength and locomotor activity), no differences in mean absolute or relative organ weights, and no overt macroscopical findings of toxicological relevance.

At 1000 mg/kg bw/day, test-item related effects included reduced mean daily food consumption in males during the initial week of treatment, and in females during the first two weeks of treatment and during the second week of gestation, reduced body weights in males throughout the treatment period and in females intermittently during pre-pairing and gestation. The mean total protein, albumin and globulin levels of the males treated with 1000 mg/kg bw/day were significantly reduced (p<0.01) when compared with the control values. These latter changes were considered to be related to the treatment with the test item. At 330 mg/kg bw/day, reduced total protein, albumin and globulin levels of females were considered to be the only findings of toxicological relevance. These changes were considered conclusive but not sufficient for the derivation of a NOAEL value.

Test item related morphological changes were noted in animals treated with 330 and 1000 mg/kg bw/day, and included increased incidence and severity of inflammatory cell infiltration in submucosa to base of lamina propria of the stomach, and considered to be a reactive inflammatory lesion (gastritis) to a repeatedly gavaged test material. The severity was slightly higher in males than females. The inflammatory change was associated with eosinophilic globule leukocytes in mucosa, and sometimes with atrophy of fundic glands, eosinophilic chief cells and epithelial vacuolation of the squamous limiting ridge at minimal to slight severity. These changes were considered to be a local effect of the test item rather than one of systemic toxicological relevance.

There were no treatment-related effects on mating performance and fertility, duration of gestation, number of corpora lutea, mean litter sizes and post natal loss. No differences on the completeness of stages or cell populations of the testes were recorded between control and high dose animals. No test item-related histological findings were recorded in the testis or in the ovary. Therefore, the NOEL/NOAEL for reprotoxicity was considered to be 1000 mg/kg bw/day, the highest dose tested; limit dose in this type of study) was derived for reproduction toxicity. This study is considered the key study.

Taking into account all available information, it is not expected that lanthanum acetate neither affect fertility nor mating performance in rats of both sexes.

Annex IX testing:

In addition to the OECD 422 study performed with lanthanum acetate, relevant studies with the read-across substance lanthanum carbonate have been performed in the framework of a drug approval (FDA, 2004). Although they do not fall under the data sharing obligations of REACH, summaries of the studies are publicly accessible and provide the most valid information for the endpoint fertility. Therefore this information is also used to support this endpoint.

Thus, a reliable one-generation fertility and embryonic development study was conducted (similar to OECD 415 guideline) in male and female Spraque-Dawley rats (SD IOPS-Caw strain) with the read-across substance lanthanum carbonate administered orally by gavage at doses of 200, 600 or 2000 mg/kg bw/day. In this study males were dosed for at least 63 days prior to mating with exposure continuing until sacrifice at the end of the mating period. Females were dosed continuously from 14 days before mating until study termination on gestational day 20.Two males of two treatment groups were found dead during the pre-mating period. One death could be attributed to a dosing error, the other could not be attributed due to cannibalism. No treatment related effects were observed on clinical signs, body weight gain, food consumption, mean number of days for mating, copulation and fertility indices for males, number of females pregnant (pregnancy rates 92, 96, 100 and 92% at 0, 200, 600, 2000 mg/kg bw/day). No treatment-related macroscopic findings were observed at necropsy. Testes weights (absolute and relative) of treated animals did not differ from those of controls. It can therefore be concluded that the NOAEL for fertility/reproductive toxicity was 2000 mg/kg bw/day, the highest dose tested in this study. A maximal reliability score of 2 (reliable with restrictions) is assigned to this study as it has been used for read-across purposes in this dossier.

In a 90-day oral repeated toxicity study (Reißmüller, 2006) in rats exposed to the read-across substance lanthanum carbonate,

no effects on male and female reproductive organs were observed

at doses up to and including 741 mg/kg bw/day for males and 1126 mg/kg bw/day for females (expressed as lanthanum carbonate anhydrous). This study was performed according to OECD 408. The study summary is included in Section 7.5.

On the basis of the results obtained in the OECD 422 test performed with lanthanum acetate, the 90-day repeated oral toxicity study (OECD 408) and the one-generation study (similar to OECD 415 guideline) performed both with the read-across substance lanthanum carbonate, it can be concluded that exposure to lanthanum does neither affect fertility nor mating performance in rats of both sexes. Furthermore, and based on the assessment of the data available, very low absorption of lanthanum is expected after exposure to lanthanum acetate (see toxicokinetic assessment in Section 7.1) and therefore low toxicity.

As no adverse effect needs to be addressed, no additional testing is scientifically justified. Therefore, and for animal welfare reasons, no further testing is proposed for lanthanum acetate. The read-across justification is included in Section 13 of IUCLID.


Short description of key information:
Toxicity to reproduction:
Braun (2013) performed a combined repeated dose toxicity study (scored K1) with reproduction/developmental toxicity screening test in rats according to OECD guideline 422 (GLP). No adverse effects were observed. A NOEL/NOAEL of 1000 mg/kg bw/day (the highest dose tested; limit dose in this type of study) was derived for reproduction toxicity. This study is considered as key study for this endpoint.

Justification for selection of Effect on fertility via oral route:
There is only one study available on lanthanum acetate. The dose of 1000 mg/kg bw/day was considered to be the NOEL/NOAEL value.

Justification for selection of Effect on fertility via inhalation route:
only one route needed for this endpoint

Justification for selection of Effect on fertility via dermal route:
only one route needed for this endpoint

Effects on developmental toxicity

Description of key information
Three studies are available: one with lanthanum acetate and two with the read-across substance lanthanum trichloride. These studies are used in a 'weight-of-evidence' approach.
Braun (2013) performed a combined repeated dose toxicity study (scored K1) with reproduction/developmental toxicity screening test in rats according to OECD guideline 422 (GLP). No adverse effects were observed. A NOEL/NOAEL of 1000 mg/kg bw/day (the highest dose tested; limit dose in this type of study) was derived for developmental toxicity.
In two publically available studies (Feng et al, 2006, Briner et al, 2000) investigating developmental effects/teratogenicity of lanthanum chloride (a 'water-soluble' lanthanum salt as lanthanum acetate) in rats and mice, the NOAEL was estimated to be 40 mg/kg bw/d corresponding to the highest dose tested.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity: oral

An OECD 422 test has been performed with lanthanum acetate. The results of the reproduction/developmental toxicity screening test indicate that lanthanum acetate is a substance of low toxicological potential, as the NOEL/NOAEL for reproductive/developmental toxicity was considered to be 1000 mg/kg bw/day (the highest dose tested, expressed as anhydrous lanthanum acetate). Furthermore no clinical signs were observed in pups, their body weight development was similar in all groups, there were no macroscopical findings of toxicological relevance and sex ratios at first litter check were considered unaffected. The study summary of this study is included in the Section 7.8.1.

In addition, two additional studies are available with the read-across substance lanthanum trichloride, a 'water-soluble' lanthanum salt as lanthanum acetate.

In a non-guideline study, groups of 10 Swiss webster mice were exposed to lanthanum chloride via drinking water at concentrations of 0, 125, 250, and 500 mg/L (corresponding to 0, 10, 20 and 40 mg/kg bw/day based on an average daily water consumption of 200 mL) 14 days prior to conception, during gestation, and until 30 days postnatally (Briner et al., 2000). Overall, litter size as well as general health and constitution including weight gain and relative brain weight did not differ significantly between the groups. However, there was a non-significant trend for delayed ear development and eye opening within exposed groups. Therefore, the NOAEL for developmental effects in this study was estimated to be 40 mg/kg bw/day, the highest dose tested.

In a non-standard study, maternal rats were exposed to lanthanum chloride from gestation day 0 through postnatal day 20 (Feng et al., 2006). From postnatal day 20, the pups were exposed to lanthanum chloride until postnatal day 150. Among other parameters, physical and neurobehavioural development of the pups was recorded. In this study, lanthanum chloride was not embryotoxic or teratogenic including the highest dose tested (40 mg/kg bw/d). Furthermore, there were no significant differences in body weight within all groups and no differences in pinna detachment and eye opening between groups. Thus, a NOAEL of 40 mg/kg bw/day was derived, which corresponds to the highest dose tested.

The results of these studies support the assumption that lanthanum is of low toxicity.

Annex IX further testing:

An OECD 422 test has been performed with lanthanum acetate. The test results indicate that lanthanum acetate is a substance of low toxicological potential, as the NOAEL for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose tested. In addition, and based on the assessment of the data available, very low absorption of lanthanum acetate is expected (see toxicokinetic assessment). Furthermore, information on the developmental toxicity after exposure to lanthanum trichloride is also available, supporting the assumption that lanthanum is of low developmental toxicity.

Additional studies are available where the developmental toxicity of the read-across lanthanum carbonate was evaluated. These studies were performed as part of a Drug application and therefore are confidential. Although no access to the studies is possible, the summaries, which are publically available in the US FDA database (2004), can be used to support the assessment of the developmental toxicity after exposure to lanthanum acetate.

In a GLP-compliant rabbit developmental toxicity study according to OECD 414 (FDA, 2004), groups of time-mated female New Zealand White rabbits received daily doses of 0 (vehicle control), 250, 750 and 1500 mg/kg bw/day of lanthanum carbonate by oral gavage from day 6 to 18 of pregnancy.The NOAEL for maternal toxicity was 750 mg/kg bw/day, while for developmental toxicity a NOAEL of 1500 mg/kg bw/day could be derived.

In a pre-postnatal study performed according to OECD 426 (FDA, 2004), time-mated female Sprague-Dawley rats were orally (gavage) exposed to lanthanum carbonate from implantation (GD 6) throughout lactation (PND 20) at doses of 200, 600, 1000 or 2000 mg/kg bw/day. The only findings that could possibly be related to maternal treatment with Lanthanum carbonate during pregnancy and lactation were observed in the high dose group and consisted of reduced body weight gain and delayed eye opening, preputial separation and vaginal opening in the F1 generation. The observed effects are often related to body weight and can probably be considered as secondary to the lower body weights in this dose group. No other landmarks of development or behavioral parameters were changed. Mating and sexual performance of the F1 generation was unaffected by the treatment. In the absence of single animal data, historical control data of the findings and of a litter based statistical analysis of the F1 generation findings no firm conclusion on the relevance of the reported effects with regard to the treatment can be drawn. A tentative NOAEL of 600 mg/kg bw/day could be derived from this study.

Based on the assessment of all available data on lanthanum acetate and other lanthanum salts, and the low absorption expected after exposure to lanthanum acetate, no further testing seems to be required for this substance as no adverse effects or hazards need to be addressed. No additional developmental toxicity test is thus proposed for animal welfare reasons, as additional testing is scientifically unjustified.

The read-across justification is included in Section 13 of IUCLID.


Justification for selection of Effect on developmental toxicity: via oral route:
An OECD 422 study including a reproduction/developmental toxicity screening is available for lanthanum acetate where the dose of 1000 mg/kg bw/day was considered to be the NOEL/NOAEL value. The robust study summary is included in endpoint 7.8.1. In addition, two studies with the read-across substance lanthanum trichloride (a 'water-soluble' lanthanum salt as lanthanum acetate) are available. No key study is selected as the three studies are used in a 'weight-of-evidence' approach.

Justification for classification or non-classification

Based on the available data and according to the DSD and CLP criteria, lanthanum acetate should not be classified as toxic to reproduction.