2-ethylhexanoic acid, cerium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined repeated dose toxicity study with reproduction/developmental toxicity screening study

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

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 sterlized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males will be 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 rurffled 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

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

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

10294-41-4

EC Number:

600-370-9

Cas Number:

10294-41-4

IUPAC Name:

10294-41-4

Details on test material:

- Name of test material (as cited in study report): cerium trinitrate
- Substance type: White crystalline
- Physical state: Solid

Test animals

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: bidistilled water

Details on oral 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.

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

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 110, 330, 1000 mg/kg
Basis:
other: Nominal concentration (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 where animals were exposed for two weeks (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:

No positive control

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, during acclimatization and up to day of necropsy.

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 and spleen.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
Males were sacrificed after treatment of 47 days, when no longer needed for the assessment of reproductive effects. Dams and pups were sacrificed on day 4 post partum. When birth did not occur on the expected date (day 21 post coitum), the dams were sacrificed on
day 25 post coitum.
All animals sacrificed in extremis or found dead 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.
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 if death occurs.
For the parent animals, special attention was directed that the organs of the reproductive system.
The number of implantation sites and corpora lutea were 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: Yes.
The tissue from all parental males was preserved in neutral phosphate buffered 4% formaldehyde solution: prostate, seminal vesicles with coagulating gland, 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 chord, small and large intestines (incl. Peyer's patches), stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroids and parathyroids if possible, trachea and lungs, uterus with vagina, urinary bladder, lymph nodes (mesenteric, mandibular), peripheral nerve (sciatic), 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 if possible tst-item related findings noted in the high dose group, the stomach and thymus of both sexes were examined from five animals in the intermediated groups.
Histological examination of ovaries on females that did not give birth (animal no. 62, 73 and 75) and the reproductive organs (testes, epididymides, prostate, seminal vesicles and coagulating glands of infertile males (animal no. 14, 25 and 27) of intermediate groups was also carried out.

A histopathology peer review was performed. The assessment of the study pathologist and reviewing pathologist compared favorably.

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 was applied 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) 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
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 WEIGHT GAIN
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.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males - pre-pairing and pairing periods:
At 1000 mg/kg/day, the mean daily food consumption values were significantly lower in males during the pre-pairing period (-39.8% (p<0.01) during days 1 - 8 and -10.8% (p<0.05) during days 8 - 13) when compared with the control males. These males largely recovered during the subsequent measurement intervals during the after-pairing period.

At 330 mg/kg/day, the mean daily food consumption values were significantly lower in males during the pre-pairing period (-11.1% (p<0.01) during days 1-8) when compared with the control males, but recovered during the remaining measurement interval of the pre-pairing period and was unaffected during the after pairing period.

The mean daily food consumption of males treated with 110 mg/kg/day compared favorably with those of the control males.

Females - pre-paring, gestation and lactation periods:
At 1000 mg/kg/day, the mean daily food consumption values were also markedly lower in females when compared with the respective controls. During days 1-8 of the pre-pairing period, the reduction (-24.2% compared to the control value) and attained statistical significance (p<0.01). This value recovered slightly during day 8-13. After being similar to control values for days 0-7 and 7-14 of gestation, the mean daily food consumption was lower (-15.5%) and attained statistical significance (p<0.05) during days 14-21. The food consumption remained lower (-23.0%) during the lactation period but was not statistically significant.

At 330 mg/kg/day, the mean daily food consumption values were similar to that of the control females.

At 110 mg/kg/day, the mean daily food consumption of the females compared favorably with the control values.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
There were minor differences in the hematology parameters, but in the absence of concomitant changes in related parameters these were considered toxicologically irrelevant.

At 1000 mg/kg/day, a small number of statistically significant differences were noted in males and females. In both sexes, slightly reduced hemoglobin levels (p<0.05) were recorded, whereas decreased hematocrit was noted only in males (p<0.01). The red cell distribution width was significantly elevated (p<0.01) in males. Increased mean absolute eosinophil count was noted (p<0.01) in males, but the relative mean eosinophil count was elevated without statistical significance. The mean platelet count was significantly elevated (p<0.05) in males when compared with the controls. Only the latter finding and the mean absolute eosinophil count exceeded the ranges of the historical control data.

At 330 mg/kg/day, the mean relative eosinophil count in males was significantly increased (p<0.05). Females showed statistically significant increases in mean relative and absolute eosinophil count (p<0.05 and p<0.01, respectively).

At 110 mg/kg/day, no differences of toxicological significance were noted.

CLINICAL CHEMISTRY
There was a small number of findings in the clinical biochemistry parameters.

In males treated with 1000 mg/kg/day, the mean globulin levels were reduced with statistical significance (19.20 g/L, p<0.05) when compared with the controls (23.75 g/L). The mean protein and albumin levels were reduced (56.27 g/L and 37.07 g/L, respectively) when compared with the control males (66.89 g/L and 43.16 g/L, respectively), but did not attain statistical significance. Females at this dose level showed significantly increased (p<0.01) triglycerides (0.69 mmol compared with 0.30 mmol/L in controls), significantly reduced (p<0.01) calcium (2.67 mmol/L compared with 2.83 mmol/L in controls), significantly decreased (p<0.01) protein (60.50 g/L compared with 72.97 g/L in controls), significantly decreased (p<0.01) albumin (42.86 g/L compared with 50.53 g/L in controls) and significantly decreased (p<0.01) globulin (17.64 g/L compared with 22.43 g/L in controls).

Females treated with 330 mg/kg/day had significantly reduced (p<0.01) level of mean protein (64.99 g/L compared with 72.97 g/L in controls) and significantly reduced (p<0.01) level of globulin (17.86 g/L compared with 22.43 g/L in controls). Albumin was reduced (47.14 g/L compared with 50.53 g/L in control) but without statistical significance.

No differences of toxicological relevance were noted at 110 mg/kg/day.

Decreased albumin levels are generally associated with insufficient nutrition and/or uptake and may correlate with the reduced body weight development.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Functional observational battery:
The mean fore-and hind limb grip strength values of the control rats were similar to those of the test item-related groups.

No test item-related findings were noted during the functional observational battery in males or females at any dose level.

Locomotor activity:
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor.
Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.

ORGAN WEIGHTS
In males treated with 1000 mg/kg/day, significantly reduced (p<0.05) testis weights (unilateral) were noted (-6.5%) when compared with the controls. In the absence of similar changes in the contralateral organ (-3.3%), these changes were considered to be incidental. The mean epididyide weight was signficantly (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 unilkely 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
Macroscopical findings:
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: NON-NEOPLASTIC
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 animals 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.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

330 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: The NOAEL for systemic toxicity of the parent animals was considered to be 330 mg/kg/day as two animals treated with 1000 mg/kg/day died as a result of the treatment-related effects in the stomach (local irritation).

2

Dose descriptor:

NOEL

Effect level:

110 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

RESULTS OF THE 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.

Applicant's summary and conclusion

Conclusions:

The NOEL (No observed effect level) for systemic toxicity of Ce(NO3)3 to the parent animals was considered to be 110 mg/kg/day, based upon the changes in the stomachs of rats treated with 330 mg/kg/day and 1000 mg/kg/day as well as differences in the mean daily food consumption/body weight development at 1000 mg/kg/day. Although these changes were considered to be related to the treatment with the test item, the morphological changes in the stomachs were considered to be local effects (irritation) rather than systemic toxicity and the differences in the food consumption/body weight were considered secondary to the changes in the stomach. However, as two animals treated with 1000 mg/kg/day died as a result of the stomach findings, the NOAEL (No Observed Adverse Effect Level) for systemic toxicity of the parent animals was considered to be 330 mg/kg/day.