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Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening test (according to OECD guideline 422; Braun, 2013), the NOAEL for reproduction toxicity was considered 330 mg/kg/day. However, it was based on effects that could be secondary to the chemical stress observed in pregnant females (due to high local irritation observed in the stomach after repeated oral gavage of the compound) rather than primary effects of the test item.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25-10-2012 to 29-04-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
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
Limit test:
no
Species:
rat
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 306 to 337 g; females: 187 to 220 g
- Fasting period before study: no data
- Housing: 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 corrected for the anhydrous active ingredient with a factor of 1.33.

The test item samples were weighed into a glass beaker on a tared Mettler balance, and part of the vehicle was added to each. Using an appropriate homogenizer, a homogeneous suspension was prepared and then the remaining vehicle was added to yield Ce(NO3)3 concentrations of 11 mg/mL, 33 mg/mL and 100 mg/mL, equivalent to 14.63, 43.89 and 133.0 mg Ce(NO3)3 x 6H2O/mL. The mixtures were then stirred with a magnetic stirrer until the test item was adequately suspended. The completed formulations were transferred to the animal room where they were again placed on magnetic stirring plates during the administration procedure to ensure homogeneous sampling. The dose formulations were prepared weekly as indicated by the results of stability analyses in the dose range-finding study.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Details on mating procedure:
During the pairing period, females were housed with sexually mature males from the same dose group (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 on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

One control female and two females at 110 mg/kg/day did not mate during the allotted 14-day pairing period. Therefore, these females were paired again but with males of their respective groups which had already successfully mated.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 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:
The cerium trinitrate peak was assigned using ion chromatographic detection in sample chromatograms by comparing to working standards. A significant cerium trinitrate peak was not detected at the retention in blank sample chromatograms, confirming that the test item was not present in the vehicle control samples (bidistilled water). The application formulations investigated during the study contained cerium trinitrate in the range of 91.4% to 103.9%. Cerium trinitrate was homogeneously distributed in the preparations and found to be stable in application formulations when stored for eight days at room temperature. The results indicate the accurate preparation of cerium trinitrate in bidistilled water as vehicle.
Duration of treatment / exposure:
Males: 47 days
Females: at least 6 weeks
Frequency of treatment:
Daily
Details on study schedule:
- First test item administration:
Males and females: day 1 of pre-pairing
- Pre-pairing:
Males and females: 14 days
-First Pairing Period:
Males and females: 14 days maximum
- Second Pairing Period:
Males and females: 3 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:
nominal conc.
(expressed as cerium trinitrate anhydrous)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a non-GLP dose range-finding study (Harlan Laboratories
Study No. D57932)

- Reserve group: 2 animals per sex. One male reserve animal was used for replacement of an animal from group 4 on the first day of treatment due
to weight loss during acclimatization as a consequence of a missing upper incisor. The remaining reserve animals as well as the replaced animal were removed from the study after commencement of the treatment.
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Animals were also observed for changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies 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 period day 1 - 8 and 8 - 13; after pairing period weekly
Females: pre-pairing period days 1-8 and 8-13; gestation days 0-7, 7-14 and 14-21, and days 1-4 of the lactation
No food consumption was recorded during the pairing period.

HAEMATOLOGY: Yes
- Collection of blood: Blood samples were obtained at the end 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 isolfurane 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 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

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
Oestrous cyclicity (parental animals):
not examined
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):
Necropsy:
All animals sacrificed in extremis were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, terminal body weights were measured and all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study when death occurred.

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.
Postmortem examinations (offspring):
Necropsy:
Dead pups, except those excessively cannibalized, were examined macroscopically.

All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study when death occurred.
Statistics:
Following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables were 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) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal
distribution
- Fischer's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Mean precoital time, fertility index and conception rate
Offspring viability indices:
Mean litter size, mean postnatal loss, rate of postnatal loss, total postnatal loss
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All males survived until scheduled necropsy. At 1000 mg/kg/day, female no 94 was found dead on day 1 of lactation and female no 95 was found dead one day later. All other females survived until scheduled necropsy.

Daily clinical signs or observations:
At 1000 mg/kg/day, test item-related clinical signs were noted during daily and detailed weekly observations in both sexes during the pre-pairing and pairing periods, consisting of ruffled fur and decreased activity. Ruffled fur was frequently noted in most females during the gestation period.

No findings of toxicological relevance were noted in males or females at 110 mg/kg/day or 330 mg/kg/day.

At 1000 mg/kg/day, ruffled fur was frequently noted in most females during the gestation period. During the lactation period, ruffled fur was noted in two females at this dose level, one of which was found dead on day 2 post partum. All other test item-treated females were unaffected.

Findings at detailed weekly clinical observations:
Males treated with 1000 mg/kg/day showed decreased activity and/or ruffled fur during the pre-pairing, pairing and after pairing periods, although the frequency of these findings decreased as the study progressed. All other test item-related males without relevant findings.

Females treated with 1000 mg/kg/day showed decreased activity and/or ruffled fur during the pre-pairing, pairing and gestation periods. As in the males, the frequency of these findings decreased as the study progressed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males - pre-pairing, pairing and after pairing periods:
In males treated with 1000 mg/kg/day, significantly lower mean body weights were noted during the pre-pairing period from days 3-14 (p<0.05 or p<0.01). The difference was -11.5% on day 14 of the pre-pairing interval. These differences continued during the pairing period (days 1 - 21, all p<0.01, amounting to -9.9% on day 21 of pairing period), and during after pairing period (days 1 - 12, p<0.01, amounting to -11.5% on day 12 of the after-pairing period) and were considered to be test item-related. The mean body weight gain values were significantly reduced from days 2-14 of the pre-pairing period (all p<0.01), significantly increased on days 6, 7 and 10 of the pairing period (all p<0.05) and significantly reduced on days 11 and 12 of the after pairing period (both p<0.05).

At 330 mg/kg/day, significantly lower mean body weights were noted during the pre-pairing period (-6.1% on day 14, day 4 and days 6-14, p<0.05 or p<0.01), and on day 1 of the after pairing period (-5.2% on day 12, p<0.05 on day 1). These differences were considered to be related to the treatment with the test item.

The mean body weights of the males treated with 110 mg/kg/day were similar to those of the respective controls. The mean body weight gain was significantly increased (p<0.05) on day 2 of pairing.

Females - pre-pairing, gestation and lactation periods:
Females treated with 1000 mg/kg/day had lower mean body weights during the pre-pairing period. These differences occurred largely between days 2-14 (-7.2% on day 14 of pre-pairing) and were statistically significant (p<0.05 or p<0.01). Lower mean body weights continued during the gestation period (-11.2% on day 21), but attained statistical significance only on days 8, 10 and 21 (p<0.05). Body weight remained lower during the lactation period (-7.2% on day 4), with statistical significance on day 3 (p<0.01) and day 4 (p<0.05). In the pre-pairing period, the mean body weight gain values showed statistically significant decreases from day 4-11 and days 13-14 (p<0.05 or p<0.01).

The mean body weights of the females treated with 330 mg/kg/day and 110 mg/kg/day were unaffected.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not examined

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm staging:
The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. There were no test item-related changes.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
During the first pairing period, one control female and two females treated with 110 mg/kg/day did not mate and in one female treated with 330 mg/kg/day mating was not detected (the body weight gain noted during days 1-13 of prepairing was 46 grams, and the female was consequently considered to be pregnant).

During the second pairing period, mating was not detected in two females treated with 110 mg/kg/day although both were found to be pregnant. The one control female which did not mate during the first pairing period mated on day 3 of the second pairing period.

One control female, one female treated with 110 mg/kg/day, two females treated with 330 mg/kg/day and two females treated with 1000 mg/kg/day were not pregnant; the fertility indices were 91.7%, 91.7%, 83.3% and 83.3%, respectively. The differences were considered to be typical biological variation. The median and mean precoital times were unaffected by treatment with the test item.

Corpora lutea count:
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups and gave no indication of a test item-related effect.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males treated with 1000 mg/kg/day, significantly reduced (p<0.05) testis weights (unilateral) were noted (-6.5%) when compared with controls. In the absence of similar changes in the contralateral organ (-3.3%), these changes were considered to be incidental. The mean epididymides weight was significantly (left and right, both p<0.05) reduced (left: -9.7% and the right: -10.4%) when compared with the control males. This was considered to be a secondary effect that resulted from the lower mean body weights. The mean brain-to-body weight ratio was significantly (p<0.01) elevated (0.54% vs 0.46%) when compared with the controls and was considered to be a secondary effect of the reduced body weights. The mean kidney-to-body weight ratio was significantly (p<0.01) elevated (0.60% vs 0.53%) but in the absence of any microscopical changes, this was considered to be unrelated to the treatment. The mean testis-to-body weight ratio (left testis only) was significantly (p<0.05) elevated (0.53% vs 0.48%) but this was considered to be due to the reduced mean body weights. Females treated with 1000 mg/kg/day showed significantly (p<0.01) elevated mean liver-to-body-weight (4.22% vs 3.60%) and significantly (p<0.05) elevated kidney-to-body weight ratios (0.61% vs 0.55%), but in the absence of any microscopical changes, this was considered to be unrelated to the treatment.

Males treated with 330 mg/kg/day were unaffected. Females treated with 330 mg/kg/day showed significantly (p<0.05) elevated mean liver-to-body weight and significantly (p<0.05) elevated liver-to-brain weight ratios (4.04% and 636.66%, respectively) when compared with the controls (3.60% and 531.05%, respectively). The latter value was dose-unrelated. This organ did not show any noteworthy microscopical changes and these differences were considered to be unlikely to be related to the treatment with the test item.

The mean absolute and relative organ weights of the males and females treated with 110 mg/kg/day were similar to those of the controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopically, dark red focus/foci of the stomach were recorded in three males treated with 330 mg/kg/day and four males treated with 1000 mg/kg/day and were considered to be test-item related. Stomach foci were not seen in females at this dose level.

All other macroscopical findings were considered to be background changes without toxicological relevance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Two females (no. 94 and 95) treated with 1000 mg/kg/day died spontaneously. Inflammatory toxic effect of the stomach and thymic atrophy at moderate severity were recorded in these animals and weakness due to the inflammatory toxic effect on pregnant animals was considered as cause of death. All other animals survived their scheduled study period.

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.
- Increase in incidence and severity of eosinophilic globule leukocyte in mucosa was recorded in animals treated with 330 and 1000 mg/kg/day.
- Atrophy of fundic gland mainly chief cells and/or parietal cells was recorded at minimal to slight severity in animals treated with 1000 mg/kg/day and males treated with 330 mg/kg/day.
- Eosinophilic chief cells were recorded at minimal to slight severity in animals treated with 330 and 1000 mg/kg/day.
- Epithelial vacuolation of squamous limiting ridge was recorded at minimal to slight severity in animals treated with 1000 mg/kg/day and males treated with 330 mg/kg/day.
Thymus:
- Increase in incidence and severity of atrophy/involution was recorded in females treated with 1000 mg/kg/day.

Other findings:
Alveolar hemorrhage and bronchioloalveolar inflammation were recorded in two males treated with 1000 mg/kg/day. However, it was considered to be an accidental lesion caused by aspiration during gavage procedures of massive amount test item. No test-item related histological findings were recorded in the ovary of females which did not give birth (animal no.: 54, 62, 73, 75, 87, and 89) and the reproductive organs of infertile males (animal no. 6, 14, 25, 27, 39 and 41). All remaining findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Dose descriptor:
NOAEL
Effect level:
330 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 generations (migrated information)
Dose descriptor:
NOEL
Effect level:
330 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 generations (migrated information)
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
Findings at first litter check and during lactation:
The mean number of pups at first litter check was not affected by the treatment with the test item. The sex ratio was also not affected. No abnormal pup was noted at any dose level.

Pup weights to day 4 post partum:
At 1000 mg/kg/day, the mean group pup weights on day 1 post partum were lower than those of the controls, and were considered to be related to the treatment with the test item. By day 4, these differences were largely compensated; the weights were largely similar to those of the respective controls.

At 110 and 330 mg/kg/day, the mean group pup weights on days 1 and 4 post partum were either similar to or greater than those of the controls.

Macroscopical findings:
At necropsy of pups, there were no abnormal findings.
Dose descriptor:
NOAEL
Effect level:
330 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Changes (esplained further) at 1000 mg/kg/day observed on day 1 post partum.
Reproductive effects observed:
not specified

Results of dose range-finding study:

- Mortality/viability:

Two of three males treated with 1888.07 mg/kg/day died before scheduled necropsy: male no. 10 was killed for ethical reasons on day 10 of treatment and male no.12 was found dead on day 6 of treatment. All other animals survived until scheduled necropsy.

- Clinical signs:

No clinical signs were evident in males or females treated with 188.77 mg/kg/day.

Ruffled fur was noted from day 2 of treatment onwards in all males treated with 566.18 mg/kg/day, and was considered to be a mild test item-related finding. Females at this dose level were unaffected.

At 1888.07 mg/kg/day, test item-related clinical signs were noted in all males and all females. In males, these findings included general weakened condition and reduced activity, hunched posture, reduced body temperature, ruffled fur (or rough coat), breathing noises, vocalization when handled, visible weight loss and blue discoloration (ie cyanosis) of the appendages. Females at this dose level were less overtly affected by the test item, yet also showed transient weakened condition, ruffled fur (or rough coat), breathing noises and visible weight loss.

- Food consumption:

At 188.77 mg/kg/day, the mean daily food consumption of the males and females compared favorably with their respective control values.

At 566.8 mg/kg/day, the mean daily food consumption values were markedly lower in males when compared with the respective controls. When compared with the control values, the mean food consumption values were -24.6%, -10.3% and -15.6% during days 1 -4, 4 -7 and 7 -14, respectively. The mean daily food consumption of the females at this dose level was similar to that of the control females.

At 1888.07 mg/kg/day the mean daily food consumption values were also markedly lower in males and females when compared with the respective controls. When compared with the control values, the mean food consumption values in males were -88.6%, -84.3% and -83.6% during days 1 -4, 4 -7, 7 -14, respectively, whereas in females the mean daily food consumption values were -82.4%, -36.2% and -10.3% during days 1 -4, 4 -7 and 7 -14, respectively.

- Body weights

The mean body weights and the mean body weight gain of the males and females treated with 188.77 mg/kg/day were similar to those of the respective controls.

The mean body weight gain of the males treated with 566.18 mg/kg/day for 14 days was lower (10.0%) than those of the control males (+19.9%). Females were largely unaffected.

The mean body weight gain of the males treated with 1888.07 mg/kg/day for 14 days were markedly lower (-30.0%) than those of the control males (+ 19.9%). Females at this dose level had lower mean body weights (+2.1%) when compared with those of the control females (+11.2%).

- Organ weights

Changes in the mean absolute and relative organ weights were largely the result of the marked loss in body weight noted in the rats treated with 1888.07 mg/kg/day.

In males, reductions of the mean absolute liver, thymus, kidney, testes and epididymides weights were considered to be related to the lower mean body weights, whereas elevated adrenal weights and elevated spleen weights may be considered stress reactions. Increased mean organ-to-body weight ratios were recorded for the brain, heart, liver, kidneys, adrenals and spleen, whereas reduced organ-to-body weight ratios were noted in the thymus, testes and epididymides. The mean brain-to-body weight ratios were accordingly decreased in the liver, thymus, kidneys, testes and epididymides and increased in the adrenals and in the spleen.

In females, a reduction of the mean thymus weight was noted. The mean liver-to-body weight was elevated and the mean thymus-to-body weight was decreased. Only the latter difference was reflected in the reduced thymus-to-brain weight ratio. All other organ weights and ratios were largely similar to those of the control females.

- Macroscopic findings

A number of findings were noted during necropsy.

At 188.77 mg/kg/day, unilateral renal pelvis dilation was noted in male no. 5, and bilateral renal pelvis distension was noted in female no. 16 and a unilateral ovarian cyst was noted in female no. 18.

At 566.18 mg/kg/day, there were no post-mortem findings in males and females.

At 1888.07 mg/kg/day, only one male (no. 11) survived until scheduled necropsy, showing size reductions of thymus, testes and epididymides. In the male which was sacrificed for ethical reasons (no. 10) on day 10 of treatment, these findings were also noted as well as stomach distension. The last male of this group (no. 12) was found dead after 6 days of treatment and showed collapsed lung (which was interpreted as a likely dosing error) and a small isolated sore on the back. The females at this dose level showed no symptoms.

Based on the results of this 14 -day dose range-finding study, dose levels of 110, 330 and 1000 mg/kg bw/day were proposed for the subsequent combined repeated dose toxicity study (with the reproduction/developmental toxicity screening test in the Han Wistar rat) with cerium trinitrate.

Conclusions:
The NOEL (No observed effect level) and NOAEL (No observed adverse effect level) for reproduction/developmental toxicity was considered to be 330 mg/kg/day, based on changes at 1000 mg/kg/day that included increased total and mean post-implantation loss, decreased mean litter size, increased mean postnatal loss, increased rate of postnatal loss, increased total postnatal loss and decreased group mean pup weights on day 1 post partum. Although such changes could be a secondary effect resulting from chemical stress observed in pregnant females (due to local irritation observed in the stomach after repeated oral gavage of the compound), a treatment-related effect could not be excluded based on the limited data available from this screening.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
330 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 (according to OECD guideline 422). Cerium trinitrate was administered to male rats for 47 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, at dose levels 0, 110, 330 and 1000 mg/kg/day (anhydrous active ingredient). Control animals were dosed with the vehicle (bidistilled water) only.

The NOAEL for reproduction/developmental toxicity was considered to be 330 mg/kg/day, based on changes at 1000 mg/kg/day that included increased total and mean post-implantation loss, decreased mean litter size, increased mean postnatal loss, increased rate of postnatal loss, increased total postnatal loss and decreased group mean pup weights on day 1 post partum. Although such changes could be a secondary effect resulting from chemical stress observed in pregnant females (due to local irritation observed in stomach after repeated oral gavage of the compound), a treatment-related effect could not be excluded based on the limited data available from this screening.

Annex IX further testing: Extended one-generation reproductive toxicity study

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test in the Han Wistar Rat study (OECD 422 guideline) has been performed with cerium trinitrate. The NOEL/NOAEL for reproduction/developmental toxicity was considered to be 330 mg/kg/day. In addition, a prenatal developmental toxicity study was performed (according to OECD guideline 414) in rat where no test item related effects were observed with the exception of body weight , body weight gain and food consumption in the high dose group at 1000 mg/kg/day. No toxicologically relevant changes on embryos or foetuses in any test item treated groups were observed. The NOAEL for maternal toxicity was established at 300 mg/kg/day; the NOAEL for embryotoxicity, foetotoxicity and teratogenicity was set at at least 1000 mg/kg/day. Based on these results, no additional effects are expected in an extended one generation reproductive toxicity study.


Effects on developmental toxicity

Description of key information

A combined repeated dose toxicity study including a reproduction/developmental toxicity screening test (OECD guideline 422; Braun, 2013) is available for cerium trinitrate. The robust study summary is included in endpoint 7.8.1. A NOAEL/NOEL for reproduction/developmental toxicity of 330 mg/kg bw/day was derived. However, it was based on effects that could be secondary to the chemical stress observed in pregnant females (due to high local irritation observed in the stomach after repeated oral gavage of the compound) rather than primary effects of the test item.
In order to address these findings observed in the OECD 422 study, a prenatal developmental toxicity study according to OECD guideline 414 was conducted.

In the OECD 414 study, cerium trinitrate was administered daily by oral gavage to pregnant Hannover Wistar rats from gestation day 6 to gestation day 19. Signs of maternal toxicity were noted in the high dose group of 1000 mg/kg/day, but there were no toxicologically relevant changes on embryos or foetuses in any of the test item treated groups. The NOAEL for embryotoxicity, foetotoxicity and teratogenicity was set as at least 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-11-02 to 2017-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar (CRL:WI(Han))
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony.
- Age at study initiation: approximately 15 weeks old at mating, young adult female rats, nulliparous and non-pregnant
- Weight at study initiation: 215-269 grams ; the variation did not exceed +/- 20% of the mean weight.
- Fasting period before study: No data
- Housing: Standard laboratory conditions; individual housing in Type II polycarbonate cages during mating and gestation period and Type III polycarbonate cages during the acclimatization period. Lignocel Hygenic Animal Bedding were used in the study. Arbocel crinklets natural nesting material was used in the study. Successfully mated animals were housed individually. Deep wood sawdust was used as bedding to allow digging and other normal rodent activities. Nest building material was also added into the cages.
- Diet (e.g. ad libitum): ad libitum, ssniff® SM Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance
- Water (e.g. ad libitum): ad libitum, tap water (in water bottles) for human consumption
- Acclimation period: At least 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4-24.9°C
- Humidity (%): 25-55%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume. During the formulation process, the pH of each formulation was adjusted using 0.1M NaOH solution to achieve a pH >= 3.5, to prevent non-specific pH damage to the stomach. The final pH values of the applied dose formulations were measured on each day of administration. Formulations were prepared fresh daily prior to administration to animals (not more than 2 hours before use) to allow their use according to the available stability information. Formulations were kept protected from light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water; based on available information of a previous rodent study and the results of a DRF study
- Concentration in vehicle: 0, 10, 30, and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg. A constant volume of 10mL/kg was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentraton and/or homogeneity was performed using a validated ICP/AES method to determine the cerium content. Top, middle and bottom samples were taken from the test item formulations two times during the study (during the first and last week of the treatment). Samples were taken in duplicate (2mL/each), one set to analyse and one set as back-up. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
Formulation samples were kept at room temperature and protected from light (in brown glass containers) until shipment to the Test Site.
Acceptance criteria of the concentration analysis was set according to the analytical method validation, at 100 +/- 15% of the nominal concentration.
Mean measured concentrations of each dose group were analyzed on 08 November 2016 and on 24 November 2016, as follows:
- 08 November analysis- 10.4 mg/mL (104% of nominal); 31.1 mg/mL (103% of nominal); 102 mg/mL (102% of nominal)
- 24 November analysis- 10.0 mg/mL (100% of nominal); 29.8 mg/mL (99% of nominal); 102 mg/mL (102% of nominal)
Details on mating procedure:
- The oaestrus cycle of female animals was examined shortly before start of pairing
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male, 1 female
- Length of cohabitation: Approximately 2 hours in the morning, daily, until at least 24 sperm-positive females/group are obtained.
- Proof of pregnancy: after the daily mating period, a vaginal smear was prepared and stained with 1% methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in vaginal smear was considered as evidence of copulation (referred to as day 0 of pregnancy, gestation day 0 or GD0). Sperm positive females were seperated and caged individually.

Duration of treatment / exposure:
from gestation day 6 to gestation day 19
Frequency of treatment:
7 days/week, daily administration, approx at similar times
Duration of test:
20 days from confirmed mating until necropsy
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control group, group 1, 0mg/mL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose group, group 2, 10 mg/mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose group, group 3, 30 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group, group 4, 100 mg/mL
No. of animals per sex per dose:
24 mated females per dose group, 4 dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were set based on available information of the chemical nature and characteristics of the test item and available results of an acute toxicity study, a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test and a dose range finding study in pregnant rats.
- In an acute oral toxicity study (OECD no. 401) in Sprague-Dawley rats, LD50 data of 4200 mg/kg was determined.
- In a combined repeated dose toxicity study with reproduction/development screening study (according to OECD guideline 422), a possible local irritation effect on stomach and mortality was observed. The NOAEL was established at 330 mg/kg/day.
- In the pregnant DRF study, concentrations up to 1000 mg/kg/day were examined using pregnant Hannover wistar rats. There were no signs of maternal toxicityother than a possible slight transient body weight difference during the first days of treatment and no embryotoxicity or foetotoxicity were observed in any test item treated dose group.
Based on this information, the doses of 100, 300 and 1000 mg/kg bw/day were deemed suitable for the purpose of the study.
- Rationale for animal assignment (if not random): The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group average of the body weight was as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.
- Justification of the strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to the experience of the Test Facility with this strain in teratology studies.
- Route of administration: the oral route is a potential route of human exposure to the test item and it was considered suitable to provide the exposure required for this developmental toxicology study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (at the beginning and end of each working day); only one general clinical observation in the afternoon on days when detailed clinical observation is made in the morning. When signs of toxicity were noted in a certain animal, this animal was observed more frequently.
- Parameters:
- signs of morbidity and mortality
- pertinent behavioural changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on all animals at the onset of treatment (GD6), then weekly. On necropsy days, detailed clinical observation was made only once.
- Parameters:
- signs of toxicity like changes in fur, skin, eyes, mucous membranes, occurence of secretions and excretions, and autonomic activity (eg 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, stereotypies (eg excessive grooming, repetitive circling), bizarre behaviour (eg self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- on GD13 and/or 14, the sperm positive females were examined for presence of vaginal bleeding or 'placental sign' (intrauterine extravasation of blood as an early sign of pregnancy in rat which is considered to confirm implantation)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.
- Corrected body weight was calculated: body weight on GD20 minus eight of the gravid uterus

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food was weighed with precision of ±1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

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

POST-MORTEM EXAMINATIONS: Yes
- Before expected delivery, on gestation day 20, Caesarean section was performed on each treated dam. Sodium pentobarbital administered by intraperitoneal injection, followed by exsanguination was used for euthanasia.
Females showing signs of premature delivery prior to scheduled necropsy will be euthanised and subjected to a thorough macroscopic examination
- Gross pathology: The dams' viscera were examined macroscopically for any structural abnormalities or pathological changes. All the gross findings were retained in 10% buffered formalin solution (or modified davidson fixative, in case of the eyes) for possible future evaluation
- Histopathology: no histopathological evaluation was performed, unless deemed necessary and depending on the effects observed.
Ovaries and uterine content:
The ovaries and uterine were removed and pregnancy status was ascertained
Examinations included:
- Gravid uterus weight: Yes, including cervix; not from animals found dead during the study. The uterus was examined for for early and late embryonic or foetal deaths and for the number of live foetuses.
- Number of corpora lutea: Yes, in each ovary
- Number of implantations: Yes , in each uterine horn
- Number of early resorptions: Yes , degree of resorption was described in order to estimate the relative time of death of the conceptus
- Number of late resorptions: Yes , degree of resorption was described in order to estimate the relative time of death of the conceptus
- Other:
- Placentas were examined macroscopically
- Uteri that appeared non-gravid were further examined to confirm the non-pregnant status
- Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss as well as foetal death.
Fetal examinations:
- External examinations: Yes; all per litter; plus an additional examination of the great arteries
- Soft tissue examinations: Yes; half of the litter
- the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in sanomya mixture then after fixation the body was microdissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.
- Skeletal examinations: Yes; half of the litter
- the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcian-blue - acetic acid - ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a diszsecting microscope.
- Head examinations: Yes; half of the litter (see above)
- Each foetus was weighed individually (accuracy +/-0.01g)
- The gender of foetuses was determined according to appearance of the anogenital distance
- All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.
Statistics:
-SAS v9.2 software package in case of Provantis v9.2:
- normality and heterogeneity of variance between groups: checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified).
- Where both tests showed no significant heterogeneity: Anova/Ancova (one-way analysis of variance) test
- If obtained result was positive, Dunnett's (Multiple Range) test to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
- If Shapiro-Wilk or Levene tests showed significance: non-parametric analysis . A Kruskal-Wallis analysis of variance was used after Rank Transformation.
-If there was a positive result: inter-group comparisons using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
-SPSS PC+4.0 program package (for data tabulated in Excel), the heterogeneity of variance between groups was checked by Bartlett's test.
- no significant heterogeneity detected: one-way analysis of variance (ANOVA)
- If obtained result significant: duncan's Multiple Range test to assess the significance of inter-group differences.
- significant heterogeneity: normal distribution of data examined by Kolmogorow-Smirnow test.
- In case of not normal distribution: non-parametric method of Kruskal-Wallis One-Way analysis of variance
- If positive result : inter-group comparisons using Mann-Whitney U-test.
- The Chi-squared test was used for comparing group incidences against the controls.
Indices:
Necropsy data
- preimplantation loss (%, group mean): (number of corpora lutea - number of implantations)/number of corpora lutea x 100
- postimplantation loss (%, group mean): (number of implantations - number of live foetuses)/number of implantations x 100
Foetal data
- sex distribution (%, group mean): (number of male(female) foetuses)/number of foetuses x 100
- external abnormalities/litter (%, group mean): (number of foetuses with abnormality)/number of foetuses x 100
- visceral abnormalities/litter (%, group mean): (number of foetuses with abnormality)/number of foetuses x 100
- skeletal abnormalities/litter (%, group mean): (number of foetuses with abnormality)/number of foetuses x 100
Historical control data:
No data
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects or systemic clinical signs were noted in the treated animals of the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) or High (1000 mg/kg bw/day) dose groups.
Slight noisy respiration (animals 3503, 3507, 3511 and 3514 in the Mid dose group; and 4510 and 4516 in the High dose group) and fur thin (animal 1510 in the Control group; and 4502 and 4513 in the High dose group) was recorded on some days, but those findings were not considered as a test item related effect.
One animal (ID 148) of the Low dose group was excluded from the study and euthanized due to scientific reason, as it delivered pups on GD 14, thus could not be used for the study purpose.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded for any animals in the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item related effect on body weight was observed in the Low (100 mg/kg bw/day) and Mid (300 mg/kg bw/day) dose groups, but an effect on body weight was noticed in the High (1000 mg/kg bw/day) dose group when compared to control.
No toxicologically significant changes were observed in the mean body weights of the Low, Mid or High dose groups when compared to control although the High dose weights remained below control until study termination.
Statistically significant differences (p<0.01) were seen in the body weight gain of the High dose group in the beginning of the treatment period (GD 6-8, GD 8-10) when compared to the control value. Although the body weight gain values recovered later, but the difference between High dose group and control (approx. 13-16%) remained statistically significant (p<0.01) when evaluated for the treatment period (GD 6-20) or entire study (GD 0-20).
Furthermore, statistically significant decreases were recorded in the corrected body weight, corrected body weight gain (GD 0-20) and net body weight gain (GD 6-20) values of the High dose group compared to the control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No biologically relevant changes were observed in the mean daily food consumption of dams in the Low (100 mg/kg bw/day) and Mid (300 mg/kg bw/day) dose group, but reduced food consumption was recorded in the High dose group (1000 mg/kg bw/day) compared to the control value.
The food consumption of the Low dose group was comparable with the Control group. Decreased food consumption (statistically significant in the Mid and High dose groups at p<0.05 and p<0.01 level, respectively) was recorded in the first half of the treatment period, although they partially recovered later. The mean daily food consumption was significantly decreased in the Mid and High dose group when calculated for the complete treatment period (by 8% and 14%, respectively). However, the difference was statistically not significant in the Mid dose group (6%) when calculated for the entire study, but it still gained statistical significance (p<0.01) in the High dose group (11%). Due to the lack of statistical significance for the study period and there was no effect on body weight or body weight gain, the observed changes in the Mid dose group were not considered as an adverse effect, in contrast to the High dose group where the observed body weight changes were seen as adverse. The major drop in food intake at the High dose group immediately after treatment initiation was a clear effect which could be expected to have the potential for secondary foetal effects.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related observations were recorded for any evaluated animals in the study.
Multifocal red discoloration of the glandular mucosa was recorded on the stomach of one high dose group animal (4523). This fur on the neck was noticed for another high dose animal (4513). However, these facts were considered as biologically not relevant findings and not related to the test item administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
No further data
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- Number of females with <=5 implantation sites: 0 in all groups
- There was no statistically significant difference in foetal death in any test item treated groups compared to the control
- The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all test item treated groups.
- No effect was observed in preimplantation loss of the test item treated groups when compared to the control
- The early and the late embryonic loss did not differ significantly from the control in the test item treated groups.
- There was no statistically significant difference in the postimplantation loss between the test item treated and control groups.
Total litter losses by resorption:
not specified
Description (incidence and severity):
Number of corpora lutea: 11.88 (control), 12.23 (low dose), 12.61 (mid dose), 12.04 (high dose)
Early or late resorptions:
not specified
Dead fetuses:
not specified
Description (incidence and severity):
- The total intrauterine mortality was comparable with the control; no statistically significant differences were observed
- There was no statitically significant difference in the number of dead foetuses in any test item treated group compared to the control
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
- 96 (24 in each group) were mated in the study.
- the number of confirmed pregnant, evaluated dams was 24 in the control group, 22 in the low dose group, 23 in the mid dose group and 24 in the high dose group
Other effects:
no effects observed
Description (incidence and severity):
Evaluation of placentas: No abnormalities were observed on the placentas of any animals in the control, low, mid or high dose groups.
Details on maternal toxic effects:
No further data
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean weight of foetuses per litter in the low, mid, and high dose groups did not differ significantly from the control mean value. In the case of the parameter 'mean body weight/foetus' significantly higher value (p<0.05) was observed in the low dose group compared to the control, but the slightly higher foetal body weight with a lack of dose response, was not considered as a test item related effect.
The total number of retarded fetuses (runts) as well as the number of affected litters was similar or lower in the test item treated group compared to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean weight of foetuses per litter in the low, mid, and high dose groups did not differ significantly from the control mean value
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The total number of retarded foetuses (runts) as well as the number of affected litters was similar or lower in the test item treated group compared to the Control group, indicating no effect on this parameter
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the external findings correspond with the current historical control or the concurrent study control data, or were considered to be incidental findings
- Number of foetuses with malformation : 0/249 (control), 1/232 (low dose), 0/245 (mid dose), 0/260 (high dose)
- Number of foetuses with variation: 2/249 (control), 0/232 (low dose), 0/245 (mid dose), 2/260 (high dose)
Two malformed foetuses were recorded in the low dose group (with craniorachischisis or with absent tail). Although no similar findigns were detected in the current study control data, but based on the isolated occurrence of both malformations and the lack of dose response, these observations were considered incidental, ascribed to individual variability and not related to treatment.
The occurence of subcutaneous haemorrhage variation was comparable with the control group data, thus these facts were considered as irrelevant findings and not related to the test item treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the skeletal findings correspond with the historical control or the concurrent study control data, or were considered to be incidental findings without dose response
- Number of foetuses with malformation : 1/123 (control), 2/116 (low dose), 2/123 (mid dose), 1/131(high dose)
- Number of foetuses with variation: 7/123 (control), 15/116 (low dose), 17/123 (mid dose), 3/131 (high dose)

- total number of intact foetuses: significantly lower in the low and mid dose groups than in the control group but no similar differences were noted in the high dose group. Similarly, the total number of foetuses with skeletal variation was slightly higher in the low and mid dose groups than in the control group but the incidence in the high dose group was comparable with the control group. due to lack of dose response, these facts were considered as no test item related effect.
- other effects, considered not related to test item treatment:
- malformation of malpositioned pelvic girdle for one foetus in mid dose group, and one foetus in control group
- other malformations as malformed or absent vertebrae (one foetus in each group) and craniorachischisis (one foetus in the low dose group)
- in case of several variations (incomplete ossification of skull, ossified sternebra, dumbbell shaped or assymmetric ossification of vertebras or way ribs, the foetal or litter based incidence in the treated groups was comparable with the current study control or historical control database
- incomplete ossification of hyoid body in 4 mid dose foetuses in two litters; no dose response
- incidence of bipartite ossification of vertebra was higher in low dose group than in current study control value , but no dose response
- incomplete ossification of tarsal bones, increased incidence observed in low and mid dose groups when compared to historical xontrol data, the foetal incidence was statistically significant in case of the mid dose group when compared to the control. However, incidence in the high dose group was similar to the historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All of the visceral findings are consistent in general nature and incidence with the study concurrent control data or the existing historical control data, therefore considered as incidental findings.
- Number of foetuses with malformation: 0/126 (control), 0/116 (low dose), 1/122 (mid dose), 0/129 (high dose)
- Number of foetuses with variation: 3/126 (control), 1/116 (low dose), 7/122 (mid dose), 17/129 (high dose)
The number of malformed/variant/intact foetuses were comparable with the control in the low and mid dose groups. In the high dose group the total number of intact foetuses were significantly lower and the number of foetuses with visceral variation was significantly higher than control. However, as these were in connection with only one variation, which was considered not being treatment related by closer special comparison with the historical control database, these facts were considered not showing a treatment related effect. In view of the maternal toxicity observed and the fact that none of the foetal visceral findings were considered to be significantly outside of the normal range, it is concluded that there was no adverse effect of the test item on the visceral observations.
No visceral malformations were recorded in any test item treated dose groups except of one malformed foetus (with total situs inversus) in the mid dose group. Based on the isolated occurrence and the lack of dose response , this observation was considered incidental, ascribed to individual variability and not related to treatment; furthermore, the historical control database contained this finding.
In case of some variations recorded in the test item treated groups (small renal papilla, convoluted ureter or dilated ureter) the calculated incidence was slightly higher than the study control or historical control values. But based on the low absolute values (only one or two litters were affected per dose group) and the lack of statistical significance, they were considered incidental findings, unrelated to test item administration.
Variation of thymic cord: in 1 low dose foetus, 4 mid dose foetuses and in 3 high dose foetuses, while only one foetus was recorded in the control group. However, there was no clear dose response, and the calcualted litter based incidences were comparable with incidences found in the historical control database for this common finding. Therefore these facts were considered as having no toxicological relevance and not being a test item related effect.
In case of short brachiocephalic trunk variation, increased incidences were observed in the mid and high dose groups compared to the control values. The increased number of foetuses with this variation in the high dose group was statistically significant compared to the control group. However, the calculated litter based incidences correlated with the frequencies seen in the historical control database. Taking into account the very slight changes and the borderline subjective judgement during foetal examination for this specific finding, the high dose was considered similar to to the upper end of the historical range, and as such it was not considered to be attributable to an adverse effect caused by the test item.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
No further data
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: absence of significant toxic effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Based on the observations made in the dams and their foetuses, signs of maternal toxicity was noted in the High dose group of 1000 mg/kg bw/day, but there were no toxicologically relevant changes on embryos or foetuses in any test item treated groups. The following no-observed-adverse-effect (NOAEL) levels were derived:
- NOAELmaternal toxicity: 300 mg/kg bw/day
- NOAELembryotoxicity: at least 1000 mg/kg bw/day
- NOAELfoetotoxicity: at least 1000 mg/kg bw/day
- NOAELteratogenecity: at least 1000 mg/kg bw/day
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

Three studies are available:

- OECD 422: Braun (2013) performed a combined repeated dose toxicity study with reproduction/developmental toxicity screening test in RCCHan: WIST (SPF) rats (according to OECD guideline 422). Cerium trinitrate was administered to male rats for 47 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, at dose levels 0, 110, 330 and 1000 mg/kg/day(anhydrous active ingredient).Control animals were dosed with the vehicle (bidistilled water) only.

The mean number of pups at first litter check was not affected by the treatment with the test item. The sex ratio was also not affected. No abnormal pup was noted at any dose level. At 1000 mg/kg/day, the mean group pup weights on day 1 post partum were lower than those of the controls, and were considered to be related to the treatment with the test item. By day 4, these differences were largely compensated; the weights were largely similar to those of the respective controls. At 110 and 330 mg/kg/day, the mean group pup weights on days 1 and 4 post partum were either similar to or greater than those of the controls. At necropsy of pups, there were no abnormal findings.

The NOAEL for reproduction/developmental toxicity was considered to be 330 mg/kg/day, based on changes at 1000 mg/kg/day that included increased total and mean post-implantation loss, decreased mean litter size, increased mean postnatal loss, increased rate of postnatal loss, increased total postnatal loss and decreased group mean pup weights on day 1 post partum. Although such changes could be a secondary effect resulting from chemical stress observed in pregnant females (due to local irritation observed in stomach after repeated oral gavage of the compound), a treatment-related effect could not be excluded based on the limited data available from this screening.

- In preparation of the prenatal development study, a dose range finding study was performed where the test item was administered via oral gavage to pregnant Hannover Wistar rats at dose levels 300, 600 or 1000 mg/kg/day. No signs of maternal toxicity other than a possible slight transient body weight difference in the first days of treatment were observed. This effect was not accompanied by a microscopic effect in the stomach. No embryotoxicity or foetotoxicity was observed in any of the test item treated groups up to 1000 mg/kg/day. This study is described as part of section 7.8.2.

- OECD 414: A prenatal development toxicity study according to OECD guideline 414 was performed (Hargitai, 2017). Cerium trinitrate was administered daily by oral gavage from gestation 6 to gestation day 19 to Hannover Wistar rats at dose levels 0, 100, 300 or 1000 mg/kg/day. All test item formulations were within the acceptance criteria of the nominal concentrations and were found to be homogenous. No test item was detected in the vehicle control samples. There were no unscheduled mortalities or clinical sings related to treatment. There were no test item related effects on maternal body weight, body weight gain, maternal corrected body weight and body weight gain, and on food intake in the low and mid dose group. In the high dose group (1000 mg/kg/day), body weight, body weight gain and food consumption were affected by test item treatment. No toxicologically significant differences in the reproductive parameters examined were observed up to 1000 mg/kg/day. There were no effects on preimplantation loss, and on the early or late embryonic loss in the test item treated dams evaluated at any dose level. The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean. The mean foetal body weight per litter was comparable with the control value in all test item treated groups. No remarkable internal or external observations were recorded for any pregnant animals during necropsy. No remarkable abnormalities were observed on the placentas in any examined groups. There were no effects of the test item on external, visceral and/or skeletal development of foetuses in the study. From these observations made in the dams and their foetuses, signs of maternal toxicity were noted in the high dose group (1000 mg/kg/day), but there were no toxicologically relevant changes on embryos or foetuses in any test item treated groups. A NOAEL maternal toxicity was set at 300 mg/kg/day based on effects on body weight (gain) and food consumption. A NOAEL for embryotoxicity, foetotoxicity and teratogenecity was set as at least 1000 mg/kg/day.

Toxicity to reproduction: other studies

Description of key information

No other studies are available

Justification for classification or non-classification

On the basis of the available information, cerium trinitrate is considered not to be classified as reproductive toxicant according to CLP regulation.

Additional information