Registration Dossier
Registration Dossier
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EC number: 909-709-8 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
In the absence of (reliable) data on the reaction mass of cerium dioxide and zirconium dioxide, the reproductive toxicity endpoints were covered using available data on (bulk) zirconium acetate (as a representative zirconium compound), bulk and nano cerium dioxide, and an organometallic form of nano cerium dioxide.
For the screening endpoint on reproductive/developmental toxicity, the following studies were included:
- An OECD 422 study performed with the 'water soluble' zirconium compound zirconium acetate, which can be considered as a representative zirconium compound allowing conclusions on the reaction mass' constituent zirconium dioxide. In this study, no effects on reproduction/development were observed up to and including at the highest dose level, resulting in NOAELs >= 1000 mg/kg bw/day.
- An OECD 422 study performed with bulk cerium dioxide (Davies, 2010a) did not observe any adverse effects on reproduction/development up to and including at the highest dose level, resulting in NOELs of >= 1000 mg/kg bw/day.
- Lee et al. (2010) performed an OECD 422 study with nano cerium dioxide and did not observe any adverse effects on reproduction/development either up to and including at the highest dose level. The NOAELs were therefore >= 1000 mg/kg bw/day as well.
- Finally, a range finding study for an OECD 416 study (2-generation reproductive toxicity study) performed with nano cerium and iron oxide isostearate according to OECD guideline 421 (Davies, 2010b) did not observe any adverse effects on reproduction up to and including at the highest dose level (i.e. NOEL >= 1000 mg/kg bw/day). At the dose level of 1000 mg/kg bw/day a lower mean body weight gain was observed in the pups between post-natal day 1 and 5, resulting in a NOEL of 450 mg/kg bw/day. Based on the results of this range finding study, 1000 mg/kg bw/day was considered suitable as highest dose level in the OECD 416 study.
Furthermore, no effect on reproductive organs were observed in repeated dose inhalation toxicity studies performed with the constituents of the reaction mass, i.e. cerium dioxide (e.g., 90 days-inhalation study in rat; Viau, 1994) and zirconium dioxide (e.g., 30-days and 60-days inhalation study in multiple animals; Spiegl et al., 1956).
For the 2-generation reproductive toxicity endpoint, an OECD 416 study performed with nano cerium and iron oxide isostearate was used as key read across study for endpoint coverage (Davies, 2010b). Both the NOAELs for the parents (parental F0/F1) and the NOELs for the offspring (offspring F1/F2) were >= 1000 mg/kg bw/day, based on the absence of adverse effects on reproductive performance and pup development at the highest dose level of 1000 mg/kg bw/day.
Taking into account all abovementioned information, it can be safely concluded that neither nanoforms neither bulk forms (should these be placed on the market) of the reaction mass of cerium dioxide and zirconium dioxide are expected to impair fertility/reproduction.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2012-12-06 to 2013-02-07
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline 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 (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 204.5 to 212.8 g (males); 164.8 to 180.2 g (females)
- Fasting period before study: none
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polisulphone solid bottomed cages measuring 59.5 x 38 x 20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polycarbonate cages measuring approximately 43 x 27 x 18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating while females were transferred to individual solid bottomed cages for the gestation period, birth and lactation.
- Diet: ad libitum, except prior to drawing of blood for clinical chemistry examinations
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15%
- Air changes (per hour): 15 to 20
- Photoperiod (hours dark / hours light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- other: purified water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS
The required amount of zirconium acetate solution (containing 40.7% of zirconium acetate anhydrous) was dissolved in the vehicle (purified water) to obtain final concentrations of 10, 30 and 100 mg/mL. The formulations were prepared daily or up to 7 days before dosing according to stability data. The concentrations were calculated and expressed in terms of zirconium acetate content (40.7%).
VEHICLE
- Concentration in vehicle: 10, 30, 100 mg/L (expressed as active compound content)
- Amount of vehicle (if gavage): 10 mL/kg body weight (for males, dose volumes were adjusted once per week for each animal according to the last recorded body weight; for females, dose volumes were calculated according to individual body weight on days 0, 7, 14 and 20 post coitum and on day 1 post partum, thereafter individual dose volumes remained constant)
- Purity: not required - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post coitum
- After 14 days of unsuccessful pairing the paired animals were separated.
- After successful mating each pregnant female was transferred to an individual solid bottomed cage for the gestation period, birth and lactation.
- Any other deviations from standard protocol: none - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (check of concentration). Samples of dosing formulations prepared on Weeks 1 and 5 were analysed to verify the concentrations. Samples of the formulations were collected and sent at ambient temperature to the analytical laboratory. Chemical analyses were carried out according to an Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) method.
- Duration of treatment / exposure:
- - Males were treated two weeks prior to pairing, throughout pairing and thereafter through the day before scheduled sacrifice (32 days of dosing).
- Females were treated two weeks prior to pairing, throughout pairing until day 3 post partum or the day before scheduled sacrifice (up to 50 days of dosing). - Frequency of treatment:
- Once daily
- Details on study schedule:
- - Age at mating of the mated animals in the study: 10 to 11 weeks
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- zirconium acetate anhydrous
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- zirconium acetate anhydrous
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- zirconium acetate anhydrous
- Dose / conc.:
- 53 mg/kg bw/day (actual dose received)
- Remarks:
- based on zirconium
- Dose / conc.:
- 159 mg/kg bw/day (actual dose received)
- Remarks:
- based on zirconium
- Dose / conc.:
- 530 mg/kg bw/day (actual dose received)
- Remarks:
- based on zirconium
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dose levels were selected in consultation with the sponsor based on information from a non-GLP 2 week preliminary toxicity study (RTC Study no. 94150EXT).
- Rationale for animal assignment: Rats were allocated to groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Rationale for selecting satellite groups: not applicable (satellite group not included)
- Post-exposure recovery period in satellite groups: not applicable (satellite group not included) - Positive control:
- None
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked each morning and afternoon for mortality.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical sign was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
- Parameters checked: The weight of food consumed by each cage of males and females.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of the sacrificial procedure
- Anaesthetic used for blood collection: yes (isofluorane)
- Animals fasted: yes
- How many animals: 5 per sex
- Parameters checked: haematocrit; haemoglobin; red blood cell count; reticulocyte count; mean red blood cell volume; mean corpuscular haemoglobin; mean corpuscular haemoglobin concentration; white blood cell count; differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells); platelets
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of the sacrificial procedure
- Anaesthetic used for blood collection: yes (isofluorane)
- Animals fasted: yes
- How many animals: 5 per sex (females with viable litters if possible)
- Parameters checked: alkaline phosphatase; alanine aminotransferase; aspartate aminotransferase; gamma-glutamyltransferase; urea; creatinine; glucose; triglycerides; bile acids; phosphorus; total bilirubin; total cholesterol; total protein; albumin; globulin; A/G Ratio; sodium; potassium; calcium; chloride
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: For males 5 days before necropsy and for females on Day 3 post partum.
- Dose groups that were examined: From each group, 5 males and 5 females were randomly selected.
- Battery of functions tested: grip strength; sensory reactivity to stimuli; motor activity assessment
FUNCTIONAL OBSERVATION BATTERY TESTS: Yes
- Time schedule for examinations: Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination.
- Battery of functions tested: removal (from cage); handling reactivity; lachrymation; palpebral closure; salivation; piloerection; rearing; spasms; myoclonia; mobility impairment; arousal (animal activity); vocalisation; stereotypies; unusual respiratory pattern; bizarre behaviour; urination; defecation; tremors; gait - Oestrous cyclicity (parental animals):
- - Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine any anomalies of the oestrous cycle.
- Sperm parameters (parental animals):
- Parameters examined in all P males:
- Testis weight, epididymis weight.
- Spermatogenic cycle: A detailed qualitative examination of the testes was performed in control and high dose groups. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. - Litter observations:
- STANDARDISATION OF LITTERS: No
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups; stillbirths; live births; postnatal mortality; presence of gross anomalies; weight gain; and physical or behavioural abnormalities.
GROSS EXAMINATION OF DEAD PUPS:
Yes, for pups surviving to day 4 post partum and for pups killed or dying during the lactation period. - Postmortem examinations (parental animals):
- SACRIFICE
- All parental animals were killed by exsanguination under isofluorane anaesthesia.
- Male animals: All surviving males were sacrificed after mating of all females was complete (after 32 days of treatment period).
- Femalel animals: Females with live pups were killed on Day 4 post partum while females which did not give birth 25 days after positive identification of mating were killed shortly (Day 27 post coitum).
TISSUE PRESERVATION: Yes
- Procedure: Samples of tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).
- Organs / tissues preserved: all abnormalities; adrenal glands; bone marrow (from sternum); brain; caecum; colon; duodenum; heart; ileum; jejunum (including Peyer’s patches); kidneys; liver; lungs (including mainstem bronchi); lymph nodes - cervical ; lymph nodes - mesenteric; nasal cavity; oesophagus; pituitary gland; prostate gland; rectum; sciatic nerve; spinal column; spinal cord (cervical, thoracic, lumbar); spleen; stomach; thymus (where present); thyroid; trachea; urinary bladder
- Reproductive organs / tissues preserved: epididymides; ovaries with oviducts; seminal vesicles with coagulating glands; testes; uterus - cervix; vagina
GROSS PATHOLOGY: Yes
- Time schedule for examinations: Terminal sacrifice. All animals.
- Organs / tissues examined: All parent animals and pups wee examined macroscopically for any structural changes.
- Reproductive organs / tissues examined: Sexual organs were specifically examined. 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 visualise possible hemorrhagic areas of implantation sites.
HISTOPATHOLOGY: Yes
- Time schedule for examinations: Tissues were collected from 5 males and 5 females (randomly selected) in the control and high dose group killed at terminal sacrifice and from all animals with abnormalities in all dose groups.
- Procedure: Tissues were dehydrated and embedded in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. Testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS) and morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
- Organs / tissues examined: all abnormalities; adrenal glands; bone marrow (from sternum); brain; caecum; colon; duodenum; heart; ileum; jejunum (including Peyer’s patches); kidneys; liver; lungs (including mainstem bronchi); lymph nodes - cervical ; lymph nodes - mesenteric; pituitary gland; prostate gland; rectum; sciatic nerve; spinal cord (cervical, thoracic, lumbar); spleen; stomach; thymus (where present); thyroid; trachea; urinary bladder
- Reproductive organs / tissues examined: epididymides; ovaries with oviducts; seminal vesicles with coagulating glands; testes; uterus - cervix; vagina
ORGAN WEIGHT: Yes
- Time schedule for examinations: Organs were collected from all animals surviving the scheduled test period.
- Procedure: Organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
- Organs / tissues examined: adrenal glands; brain; heart; kidneys; liver; prostate gland; spleen; thymus
- Reproductive organs / tissues examined: epididymides; ovaries with oviducts; testes - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring surviving to post partum Day 4 and pups killed or dying during the lactation period were sacrificed on post partum Day 4.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY
- All pups found dead in the cage were examined for external and internal abnormalities.
- All live pups sacrificed at termination were examined for external abnormalities.
HISTOPATHOLOGY / ORGAN WEIGHTS
Examinations not performed - Statistics:
- - Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
- Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
- The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
- The criterion for statistical significance was p<0.05 - Reproductive indices:
- - The following reproductive indices were calculated: copulatory index; fertility index; pre-coital interval (mean number of days between pairing and mating); pre-implantation loss and pre-birth loss
- Offspring viability indices:
- - The following viability indices were calculated: pup loss at birth and cumulative pup loss on Day 4 post partum
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- (toxicologically irrelevant)
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- (reproductive effects)
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Remarks:
- anhydrous zirconium acetate
- Sex:
- male/female
- Basis for effect level:
- other: Based on a lack of toxicologically relevant effects on reproductive organs/tissues or reproductive performance of parental animals.
- Critical effects observed:
- no
- 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
- Dose descriptor:
- NOAEL
- Remarks:
- (developmental effects)
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- act. ingr.
- Remarks:
- anhydrous zirconium acetate
- Sex:
- male/female
- Basis for effect level:
- other: Based on a lack of developmental effects on pups in any dose group.
- Critical effects observed:
- no
- Reproductive effects observed:
- no
- Conclusions:
- No effects on reproduction or development were observed in any dose group or in pups. Therefore, on the basis of the results obtained in the study, the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity and for developmental toxicity was considered to be >=1000 mg/kg bw/day (expressed as zirconium acetate anhydrous).
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- April 2008 - August 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline 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 (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 10 weeks old
- Weight at study initiation: 402 - 454 g (males) / 244 - 285 g (females)
- Fasting period before study: no
- Housing: individual (except during pairing), in wire-mesh cages (43 x 21.5 x 18 cm) or polycarbonate cages (43 x 21.5 x 20 cm) for females during lactation
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20°C
- Air changes: about 12 per hr
- Photoperiod: 12 hrs dark / 12 hrs light (7.00 am - 7.00 pm)
IN-LIFE DATES: From 14 May 2008 To 14 July 2008 - Route of administration:
- oral: gavage
- Vehicle:
- other: 0.5% aqueous methylcellulose solution
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Test item ground to fine powder using mortar and pestle, suspended in vehicle and homogenized by magnetic stirrer
- Dosing solutions prepared for use for up to 12 days and stored in brown flasks at +4°C, protected from light, prior to use
VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate for oral suspensions
- Concentration in vehicle: 30, 90 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): Sigma batches 017K0052 and 066K0129 (methylcellulose)
- Purity: 0.5% - Details on mating procedure:
- - M/F ratio per cage: one female was placed with one male
- Length of cohabitation: Each female was placed with the same male until mating occurs or 14 days had elapsed. Any pairs with no evidence of mating after 14 days were separated and the female was placed for 7 days with a different male from the same dose level group who had already mated.
- Proof of pregnancy: Confirmation of mating was made in the morning, every day up to proof of mating, by checking for the presence of a vaginal plug or of sperm in a vaginal lavage.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- During the pre-study period, the homogeneity, stability and concentration of two dosage forms prepared at low and high concentrations (33.6 and 230 mg/mL) were checked using ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry) after validation of the analytical method. The results showed acceptable homogeneity and stability of both concentrations over 12 days of storage at +4°C protected from light.
During the study, the concentration of the test item (0, 33.6, 101.7 and 230 mg/mL in week 1 and 0, 30, 90 and 200 mg/mL in week 6) and homogeneity of the dosage forms were determined in samples of each control and test item dosage form prepared for use in weeks 1 and 6. The results showed acceptable homogeneity and concentration of all dosage forms analyzed. Precision (RSD ≤ 10%) and accuracy (100 ± 10%) of the method were found to be satisfactory. The homogeneity of the dosage form prepared for week 6 at 200 mg/mL was slightly outside the acceptance criteria with a RSD of 12.8% but this was considered to have no impact on the validity of the study. - Duration of treatment / exposure:
- - Males: 15 days before mating, during mating (up to 3 weeks), until euthanasia (4 weeks total)
- Females: 15 days before mating, during mating (up to 3 weeks), during pregnancy, during lactation, until day 5 post partum inclusive - Frequency of treatment:
- Once daily, 7 days a week
- Details on study schedule:
- Not appropriate
- Dose / conc.:
- 0 mg/kg bw/day
- Dose / conc.:
- 168 mg/kg bw/day (nominal)
- Remarks:
- Basis: nominal, up until day 3 due to an error of density measurement
- Dose / conc.:
- 509 mg/kg bw/day (nominal)
- Remarks:
- Basis: nominal, up until day 3 due to an error of density measurement
- Dose / conc.:
- 1 150 mg/kg bw/day (nominal)
- Remarks:
- Basis: nominal, up until day 3 due to an error of density measurement
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Remarks:
- Basis: nominal from day 4 on
- Dose / conc.:
- 450 mg/kg bw/day (nominal)
- Remarks:
- Basis: nominal from day 4 on
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Basis: nominal from day 4 on
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: on the basis of a previous 10-day dose-range finding study (CIT No. 33178 TSR) at the same dose levels
- Rationale for animal assignment (if not random): computerized stratification procedure so that the average body weight of each group was similar - Positive control:
- Not included
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (morbidity and mortality) or once daily (clinical signs)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before beginning of treatment period and once weekly during treatment period
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.
BODY WEIGHT: Yes
- Time schedule for examinations:
Males: on first day of treatment and then once weekly
Females: on first day of treatment and then once weekly until mated, and on days 0, 7, 14 and 20 post coitum and days 1 and 5 post partum
FOOD CONSUMPTION: Yes
- Time schedule for examinations:
Males: once weekly over 7-day periods from first day of dosing
Females: once weekly over 7-day periods from first day of dosing through gestation and lactation
Not recorded during pairing period
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight for at least 14 hours)
- How many animals: first 5 males and 5 females to deliver in each group
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology, reticulocytes, prothrombin time, activated partial thromboplastin time, fibrinogen
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight for at least 14 hours)
- How many animals: first 5 males and 5 females to deliver in each group
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, bile acid
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of treatment period (day 5 post partum for females)
- Dose groups that were examined: all groups (first 5 males and 5 females to deliver)
- Battery of functions tested: sensory activity / grip strength / motor activity / other: standard reflexes and responses, rectal temperature
Details:
The FOB included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity. The animals were randomized in order to ensure "blind" evaluation. All animals were observed in the cage, in the hand and in the standard arena.
The following parameters were assessed and graded:
- "touch escape" or ease of removal from the cage,
- in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Then, the following parameter measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupillary reflex,
- visual stimulus response,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.
OTHER: No other general toxicity parameter - Oestrous cyclicity (parental animals):
- Estrous cyclicity determined from a fresh vaginal lavage (stained with methylene blue) every morning during mating period until effective mating
- Sperm parameters (parental animals):
- Microscopic examination made special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in offspring:
- Stillbirths, live births
- The total litter size and numbers of pups of each sex (sex ratio) was recorded as soon as possible after birth.
- Postnatal mortality: The litters were observed daily in order to note the number of live, dead and cannibalized pups.
- The pups were observed daily for clinical signs.
- Weight gain: The weight of each pup was recorded on days 1 and 5 post-partum.
GROSS EXAMINATION OF DEAD PUPS: yes (external abnormalities) - Postmortem examinations (parental animals):
- SACRIFICE
After overnight fasting, at least 14 hours, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
- males: after the end of the mating period (after at least 4 weeks of treatment),
- females: on day 6 post-partum,
GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were also recorded.
HISTOPATHOLOGY / ORGAN WEIGHTS
A microscopic examination was performed on:
- all the tissues listed in the Table below for the first five males and females of the control and high-dose groups (groups 1 and 4) sacrificed as scheduled
- all macroscopic lesions
The microscopic examination made special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. - Postmortem examinations (offspring):
- Pups found dead, prematurely sacrificed and pups sacrificed on day 5 post-partum were carefully examined externally for gross external abnormalities and a macroscopic examination was performed. There was no preservation of tissues.
- Statistics:
- - Comparison of mean values by one-way variance analysis and Dunnett's test
- Percentage values compared by Fisher's exact probability test
- Specific sequences of tests used for clinical chemistry and organ weight data - Reproductive indices:
- The following parameters were evaluated:
- Pre-implantation loss,
- Post-implantation loss,
- Mating index,
- Fertility index,
- Gestation index,
- Number of corpora lutea,
- Number of implantations,
- Number of pups delivered - Offspring viability indices:
- The following parameters were evaluated:
- Live birth index,
- Viability index on day 4 post-partum,
- Mean litter size,
- Pup sex ratios - Clinical signs:
- no effects observed
- Description (incidence and severity):
- Isolated and non-dose-related incidences of soft feces, loud breathing, chromorhinorrhea and reflux at dosing were observed in all groups treated with Cerium oxide. Generally only one animal was affected and the signs were short-lived. It was considered that none of these represented signs of toxicity of the test item.
In addition, hairloss and cutaneous lesions were observed in the control group and the groups treated at 150 or 450 mg/kg/day. These signs are commonly observed in laboratory rats of this strain and are considered not to be related to treatment with the test item. - Mortality:
- no mortality observed
- Description (incidence):
- There were no unscheduled deaths during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on mean male or female body weight or body weight gains during the premating, gestation or lactation phases.
The female group treated at 450 mg/kg/day had a slightly lower mean body weight gain over the gestation period when compared with the controls but the difference was mainly due to one female with a low body weight gain which skewed the group mean. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on mean male or female food consumption.
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- A detailed, stage-aware qualitative evaluation of the testes was conducted in control and high dose males.
There were no microscopic findings related to the test item administration.
In the few low-, mid-, and high-dose females which were sacrificed because of no delivery, no estrous cycle abnormalities were found at microscopic examination of the genital organs. However, in high-dose female R24319, a congenital anomaly involving the cervix and distal vagina explained the absence of mating/pregnancy. Both the cervix and the distal vagina were dilated with presence of a large mucosal protrusion in the cavity. - Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on mating; all females at all dose-levels mated and only one male treated at 150 mg/kg/day did not mate.
The mean number of days of pairing before mating was within the normal range, although the groups treated at 150 or 450 mg/kg/day took longer to mate than the controls; up to 4 or 5 days of pairing be fore mating is considered to be normal based on a normal estrous cycle of 4 to 5 days.
Females R24297 (150 mg/kg/day) and R24303 (450 mg/kg/day) stayed for 1 week or more in diestrus before mating. Both females were not pregnant. Other than these two females, all estrous cycles were considered to be normal.
There were two, one and three non-pregnant females in the groups treated at 150, 450 or 1000 mg/kg/day, respectively. In the non-pregnant female R24319 (1000 mg/kg/day), a congenital anomaly involving the cervix and distal vagina explained the absence of mating/pregnancy. Females R24315 and R24316 (1000 mg/kg/day) were in pro-estrous on the day before mating and so should have been in estrous on the day of mating. Females R24303 (450 mg/kg/day) and R24297 (150 mg/kg/day) had been in diestrous for longer than normal before mating but this is not uncommon and pregnancy does occur after a prolonged period of di-estrous. Female R24291 (150 mg/kg/day) mated on the first day so the cycle stage was unknown. The last three females may have mated on an inappropriate day in the estrus cycle which could explain the lack of pregnancy. Although non-pregnancy was not observed in control females and no sound explanation was found for all non-pregnant females but one, the absence of pregnancy in females treated at 150, 450 or 1000 mg/kg/day was considered in
cidental and not related to the test substance.
There were no effects on the mean numbers of corpora lutea, implantations or pups at any dose level, nor on the duration of gestation or the extent of post-implantation loss. The mean pre implantation loss was higher in all groups treated with the test item when compared with the controls, however these variations were not dose-related nor statistically significant and there was no influence on the mean number of pups delivered betwwen control and treated groups. It was therefore considered that this did not represent an effect of treatment with the test item. - Dose descriptor:
- NOEL
- Remarks:
- mating and fertility
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other: no relevant effects up to highest dose tested
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Isolated clinical signs were observed in a few pups treated at 150 mg/kg/day, typically hematoma or scabbing. It was considered that there were no effects of treatment with the test item.
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- Pup survival was similar between the control group and the groups treated with Cerium oxide.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item; the mean body weights and mean body weight gain of the pups from the groups treated with test item were slightly higher than those of the controls on days 1 and day 5 post-partum.
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- One pup from the group treated at 450 mg/kg/day had malpositioned right testis, small right epididymis and pale right kidney. As these were observed in only one pup out of 137 in this group and out of a total of 346 pups from the groups treated with test item, it was considered not to be related to treatment but to be a congenital malformation.
- Histopathological findings:
- not examined
- Dose descriptor:
- NOEL
- Remarks:
- toxicity on progeny
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- act. ingr.
- Sex:
- male/female
- Basis for effect level:
- other: no relevant effects up to highest dose tested
- Reproductive effects observed:
- not specified
- Conclusions:
- No significant reproductive or developmental toxicity was observed up to the highest tested dose, i.e. 1000 mg/kg.
- Executive summary:
In an OECD TG 422-compliant study, the potential general and reproductive or developmental toxicity of Cerium Oxide were tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0 (0.5% aqueous methylcellulose solution), 150, 450 or 1000 mg/kg (except for the 3 days of dosing when an error in density measurements resulted in dose levels of slight overdosing at 168, 509 or 1150 mg/kg, respectively).
No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects on body weight, body weight gain or food consumption at any dose level. The Functional Observational Battery assessment, hematology and blood chemistry parameters revealed no treatment-related effects. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters. Pups showed no effects of treatment on survival or body weight performance. Macroscopic and microscopic examinations at necropsy did not reveal any treatment-related findings and there were no treatment-related changes in organ weights.
The NOELs for parental toxicity, for reproductive performance (mating and fertility) and for toxic effects on progeny were therefore all considered to be >= 1000 mg/kg.
No classification for repeat-dose toxicity or reproductive or developmental toxicity is warranted based on the absence of relevant effects in this study, according to the criteria of Annex VI Directive 67/548/EEC or UN/EU GHS.
This study is classified as acceptable. It satisfies the OECD 422 guideline requirements on repeated dose toxicity testing and reproduction/developmental toxicity screening.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- from 23 May 2008 to 15 April 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The study was performed similarly to OECD guideline 421 and following the principles of Good Labaratory Practices (but not audited by Quality assurance unit). This study was used as a preliminary test for a 2-generation study.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- only 2 dose levels, no microscopic examination was performed, used as a preliminary study to a multigeneration study (CIT study n° 34760 RSR)
- GLP compliance:
- no
- Remarks:
- but followed the principles of Good Laboratory Practices
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: mean body weight of 427 g (range: 413 g to 444 g) for the males and 266 g (range: 251 g to 292 g) for the females.
- Fasting period before study: no
- Housing: individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). Towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet.
- Water: free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: 6 days.
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: about 12 cycles/hour
- Photoperiod: 12hrs dark / 12hrs light
IN-LIFE DATES: from 23 May 2008 (arrival of the animals) to 27 July 2008 (necropsy of the last female) - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle.
The test item was mixed with the required quantity of vehicle and then passed in an ultraturax for at least 5 minutes and until the consistency appeared acceptable in order to achieve the concentrations of 90 and 200 mg/mL. The resulting suspension was left under magnetic stirring until treatment of the animals.
VEHICLE
- Justification for use and choice of vehicle: Vehicle used in previous repeated (28-day) dose toxicity study (22314 TSR) in which stability, homogeneity and concentration of the test item in the vehicle were found satisfactory.
- Concentration in vehicle: 90 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
- Lot/batch no.: 126K0117 and 117K0127 - Details on mating procedure:
- - M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually - Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- No chemical analysis was performed.
- Duration of treatment / exposure:
- - In the males: 15 days before mating, during the mating period (up to 3 weeks), until sacrifice (i.e. at least 5 weeks in total).
- in the females: 15 days before mating, during the mating period (up to 3 weeks), during pregnancy, during lactation until day 4 post-partum inclusive. - Frequency of treatment:
- Once a day, at approximately the same time each day, 7 days a week.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 450 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10 males and 10 females per group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: the dose-levels were selected on the basis of a 4-week toxicity study in the rat (CIT/Study No. 22314 TSR). In this study, dose-levels of 150, 450 and 1000 mg/kg/day resulted in no deaths or clinical signs, no relevant effects on body weight or food consumption, no toxicologically significant differences in hematology or blood biochemistry parameters and no effects were observed at microscopic examination. The NOEL was considered to be 1000 mg/kg/day.
- Rationale for animal assignment: according to a computerized stratification procedure, so that the average body weight of each group was similar. - Positive control:
- not required.
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day for mortality and morbidity including week-end and public holidays. Once a day for clinical signs.
- Cage side observations include routine examinations.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations:
> for males: the first day of treatment (day 1), then once a week until sacrifice;
> for females: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.
FOOD CONSUMPTION:
The quantity of food consumed by each male was recorded once a week, over a 7-day period, from the first day of treatment until sacrifice.
The quantity of food consumed by each female was recorded once a week, over a 7-day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice.
During the pairing period, food consumption was not recorded for males or females.
WATER CONSUMPTION: No - Oestrous cyclicity (parental animals):
- The estrous cycle stage was determined from a fresh vaginal lavage, each morning during the mating period, until the females had mated.
- Sperm parameters (parental animals):
- Not examined.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter size and number and sex of pups, stillbirths, live births, dead and cannibalized pups, postnatal mortality, presence of gross anomalies, clinical signs (daily), body weight (day 1 and day 5 post-partum, weight gain, physical or behavioural abnormalities
GROSS EXAMINATION OF DEAD PUPS:
Pups found dead or prematurely sacrificed were carefully examined for gross external abnormalities. Pups sacrificed on day 5 post-partum were discarded with no post-mortem examination. There was no preservation of tissues. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals , after the end of the mating period (after at least 5 weeks of treatment).
- Maternal animals: All surviving animals, on day 5 post-partum, or on day 25 post-coitum for females which had not delivered.
GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation scars were also recorded for females sacrificed on day 5 post-partum.
The numbers of corpora lutea were recorded for females sacrificed on day 25 post-coitum due to no delivery. For these apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
HISTOPATHOLOGY / ORGAN WEIGHTS
All macroscopic lesions were preserved in 10% buffered formalin (except for the testes and epididymides which were preserved in Davidson’s fixative).
No microscopic examination was performed. - Postmortem examinations (offspring):
- - Pups were sacrificed on day 5 post-partum.
- Pups found dead or prematurely sacrificed were carefully examined for gross external abnormalities. Pups sacrificed on day 5 post-partum were discarded with no post-mortem examination.
- There was no preservation of tissues. - Statistics:
- Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by the Fisher exact probability test. - Reproductive indices:
- The following parameters were evaluated:
- Pre-implantation loss,
- Post-implantation loss,
- Mating index,
- Fertility index,
- Gestation index,
- Number of corpora lutea,
- Number of implantations,
- Number of pups delivered - Offspring viability indices:
- The following parameters were evaluated:
- Live birth index,
- Viability index on day 4 post-partum,
- Mean litter size,
- Pup sex ratios - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A total of three animals treated at 1000 mg/kg/day showed relevant clinical signs: one male showed ptyalism from day 12 to day 26 of dosing, one female showed reflux at dosing on day 12 post-coitum only and another female displayed chromodacryorrhea on day 21 post-coitum and day 0 post-partum.
One male treated at 450 mg/kg/day showed reflux at dosing on day 9 of dosing.
It was considered that none of these represented signs of toxicity of the test item.
In addition, hairloss on the forelimbs was observed in one control female, and one male and one female treated at 1000 mg/kg/day. These signs are commonly observed in laboratory rats of this strain and are considered not to be related to treatment with the test item. - Mortality:
- no mortality observed
- Description (incidence):
- There were no unscheduled deaths during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on males or females during the pre-mating phase.
During gestation, females treated at 1000 mg/kg/day gained more weight than the controls but the females treated at 1000 mg/kg/day had a mean of one pup more than the control females which explain the higher body weight gain.
Both female groups treated with the test substance gained more body weight than the controls during lactation, however four control females lost a small amount of weight during this period. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on mean male or female food consumption.
The female group treated at 1000 mg/kg/day had slightly higher food consumption than the controls during lactation which corresponded with the higher body weight gain. - 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
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- 1 control female and 2 females at 450 mg/kg/day stayed for 1 week or more in diestrus and/or metestrus before mating; all females were pregnant. Other than these females, all estrous cycles were considered to be normal.
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on mating; all females at all dose-levels mated and only one control male did not mate. The mean number of days of pairing before mating was similar to or lower than the control group.
There were two mated but non-pregnant females in the group treated 1000 mg/kg/day. Neither showed any macroscopic abnormality at post-mortem examination.
There were no effects on the mean numbers of corpora lutea, implantations or pups at any dose-level, nor on the duration of gestation or the extent of pre-implantation loss.
The mean post-implantation loss was slightly higher in the groups treated with the test item when compared with the controls, however the differences were not toxicologically significant. It was therefore considered that this did not represent an effect of treatment with the test item.
Sex ratio: The percentage of male pups was similar between all groups. - Dose descriptor:
- NOEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No relevant effects up to highest dose tested
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs were observed at 1000 mg/kg/day. At 450 mg/kg/day, one pup was cold to the touch, showed generalized hematoma and was found dead on post-natal day 4. Another pup from the same litter appeared to have milk in the urogenital region. It was considered that there were no treatment-related clinical signs.
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- Pup survival was higher in the groups treated with the test substance than in the control group.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The mean body weight on post-natal day 1 was similar between all groups, including the controls, but on post-natal day 5 the pups from the group treated at 1000 mg/kg/day had a lower, non-statistically significant, mean body weight than the controls (-7% in the males and -6% in the females). The mean body weight gain from post-natal days 1 to 5 was also lower at 1000 mg/kg/day (-15% in the males and -12% in the females) when compared with the controls. This may have some relationship to the fact that there was a mean of one pup more per female in this group because the individual values at 1000 mg/kg/day were within the control range, but a relationship to treatment with the test item cannot be excluded.
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- None of the pups found dead showed gross external abnormalities.
- Histopathological findings:
- not examined
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 450 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Critical effects observed:
- no
- Reproductive effects observed:
- no
- Conclusions:
- Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.
- Executive summary:
In a Reproduction/developmental toxicity screening test, the potential general toxicity and reproductive or developmental toxicity of the test item were tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0, 450 or 1000 mg/kg/day in corn oil.
No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.
Pups showed no effects of treatment on survival.
Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg/day (-15% in the males and -12% in the females, not statistically significant). This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded. It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio. There were no treatment-related macroscopic abnormalities.
Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.
As this study was designed to choose the dose-levels which should be used in a 2-genarations study, it is however considered that 1000 mg/kg/day would be a suitable high dose-level for the multigeneration study.
This study is classified as acceptable. It is similar to OECD guideline on reproduction/developmental toxicity screening and is acceptable as a preliminary study to a multigeneration study.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2020
- 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)
- Version / remarks:
- 0ECD 1996
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Orient Bio, Inc. (Republic of Korea)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks of age
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 1 or 2 per stainless-steel cage (255W × 465L × 200H mm3). Pregnant and lactating dams were housed individually in a poly-sulfone cage (260W × 420L × 180H mm3) with sterilized Aspen animal bedding (Bio Lab, Republic of Korea) during the study period.
- Diet (e.g. ad libitum): The sterilized commercial rodent feed (PMI Nutrition International, USA) was also provided ad libitum.
- Water (e.g. ad libitum): The water was irradiated by UV light and filtered prior to provide ad libitum.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C,
- Humidity (%): 50 ± 20%,
- Air changes (per hr): 10–20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light-dark cycle
IN-LIFE DATES: The experimental phase of this study was conducted in 2015. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- CeO2 NPs were diluted in deionized water and sonicated by the Vibra-Cell® sonifier with a 13 mm probe at 25% amplitude for 8 min. Dose formulations were mixed by a stirrer during the dosing, and dosing volume was 10 ml/kg.
- Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks.
- Proof of pregnancy: Mating was confirmed by the presence of sperm in the vaginal smear and/or the vaginal plug, and this was considered GD 0.
- After successful mating each pregnant female was caged (how): individually - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- - Males were administered during a 2-week premating period and during mating and up to the final sacrifice in males (total of 38 days).
- Females were administered during a 2-week premating period and during mating, gestation and up to lactation day (LD) 4(total of at least 41 days). - Frequency of treatment:
- daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Negative control : deionized water (vehicle)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 12 males and 12 females
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose levels were selected on the basis of the results of a preliminary study with CeO2 NPs in SD rats (5 animals/sex/group). Animals were daily dosed CeO2 NPs with 100, 300 and 1000 mg/kg dose levels for two weeks prior to mating, and dosing was continued through final sacrifice in males (total 28 days) and through gestation day (GD) 15 in females (total of at least 29 days).
There was no test item-related change in all examined parameters, including clinical signs, body weight, food consumption, clinical pathology, macroscopic observation, organ weights, fertility, and cesarean section, at any doses tested. Therefore, 1000 mg/kg, which is the limit dose level, was selected as the high dose, and 300 and 100 mg/kg were determined to be the intermediate and low doses, respectively.
- Rationale for animal assignment (if not random):
Healthy animals with adequate body weight increase and exhibiting no clinical signs were used in this study. Twelve male and twelve female SD rats were divided to each of the groups to have a similar mean body weight using the Pristima system (Xybion Medical System Co., USA).
- Fasting period before blood sampling for clinical biochemistry: Yes, approximately 16 hours (overnight) prior to sacrifice - Positive control:
- no
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical examinations including mortality and general clinical signs were examined twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs were examined once weekly during the study period
BODY WEIGHT: Yes
- Time schedule for examinations: Animal body weights were measured twice weekly during the pre-mating and mating periods. Mated females were weightedon days 0, 7, 14 and 20 of gestation, and on days 0 and 4 of lactation.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured in the same days of the body weight measurement except for the mating and was calculated as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified.
OTHER:
> FOB :
Functional observations of animals, including sensory function tests (tail pinch, approach and touch response, pupillary reflex and acoustic startle response), grip strength and motor activity, were conducted with 6 animals/sex/group before necropsy (Moser 1991; Pierce and Kalivas 2007).
> Hematogoly and chemical chemistry :
- Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice.
- Blood for clinical pathology were collected from the caudal vena cava from 5 randomly
selected animals/sex/group. Blood for hematology was placed into tubes containing potassium salt of ethylenediaminetetraacetic acid (EDTA) and then analyzed with an ADVIA2120i hematology analyzer (Siemens, Germany) for the following parameters: total red blood cell count (RBC), mean corpuscular volume (MCV), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), platelet count (PLT), reticulocyte count, total white blood cell count (WBC) and WBC differential count (absolute and relative counts of neutrophils [NEU], lymphocytes [LYM], monocytes [MON], basophils [BAS] and eosinophils [EOS]). Blood for coagulation was put into tubes containing 3.2% sodium citrate and centrifuged (approximately 3,000 rpm, 10 min, at room temperature) to obtain plasma. A coagulation test was conducted with an ACL 9000
coagulation analyzer (Instrumentation Laboratory, Italy) for the following parameters: activated partial thromboplastin time (APTT) and prothrombin time (PT).
- Blood samples for clinical chemistry were placed into tubes without anticoagulant and kept at room temperature for a minimum of 90 min and then centrifuged (approximately 3000 rpm, 10 min, at room temperature) to obtain serum. Clinical chemistry analysis was conducted with a Toshiba 200 FR NEO chemistry analyzer (Toshiba Co., Japan) for the following parameters: glucose (GLU), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), alkaline phosphatase (ALP), total cholesterol (TCHO), triglyceride (TG), albumin/globulin ratio (A/G), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA), phospholipid (PL), creatine phosphokinase (CK), sodium (Na), inorganic phosphorus (IP), calcium (Ca), potassium (K) and chloride (Cl). - Oestrous cyclicity (parental animals):
- Oestrous cyclicity was measured.
- Sperm parameters (parental animals):
- All male reproductive organs (testes, epididymides, seminal vesicles with coagulation glands and prostate) were weighed and processed for histopathological analyses.
- Litter observations:
- PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities.
- Postmortem examinations (parental animals):
- SACRIFICE
All surviving males on the day after final dosing and females on LD 5 were humanely sacrificed with isoflurane. Blood for clinical pathology were collected from the caudal vena cava from 5 randomly selected animals/sex/group. Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice.
GROSS NECROPSY
All animals were subjected to macroscopic observations.
HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were examined and preserved in 10% neutral buffered formalin or an appropriate fixative for histopathology: ovaries, testes, uterus with cervix, brain, stomach, ileum, duodenum, jejunum, colon, cecum, rectum, liver, kidneys, adrenal glands, spinal cord (cervical, thoracic, lumbar), prostate, epididymides, seminal vesicles with coagulation glands, thyroid with parathyroid glands, trachea, lungs with bronchi, mesenteric lymph nodes, mandibular lymph nodes, urinary bladder, femur with marrow, sciatic nerve, spleen, heart, thymus and abnormal lesions. All reproductive organs and the other organs from 5 animals per sex in each group were further processed to slides and stained with hematoxylin and eosin for histopathological examinations. Kidneys were also examined in the low- and intermediate-dose groups to further investigate the treatment-related changes.
All male reproductive organs (testes, epididymides, seminal vesicles with coagulation glands and prostate) were weighed, and the following organs were weighed from 5 animals per sex in each group: liver, kidneys brain, pituitary gland, heart, thymus, spleen, ovaries, adrenal glands, lungs and uterus with cervix. Paired reproductive organs were weighed separately. - Postmortem examinations (offspring):
- After parturition, pup mortality and general clinical signs were examined once daily. Pup external abnormalities were recorded. Pup individual body weight and sex were recorded on post-natal day (PND) 0 and 4, and these data were reported for each litter.
- Statistics:
- Statistical analyses were conducted based on the general statistical method used in this type of toxicology study and our previous study (Lee et al. 2019). Statistical analysis was performed using the Pristima System or Statistical Analysis Systems (SAS Institute, USA), and the level of significance was taken when p < 0.05 or p < 0.01. The litter was used as a statistical unit for litter data.
Pup body weight was analyzed using one-way analysis of covariance (ANCOVA), and the litter size was used as the covariate. - Reproductive indices:
- > Based on these mating results, the number of days the animals were confirmed to mate (precoital time) and fertility-related data, including mating, fertility, fecundity and pregnancy index, were calculated.
> The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups. - Offspring viability indices:
- > After parturition, pup mortality and general clinical signs were examined once daily. Based on the parturition and pup mortality results, the delivery index (% of
dams with live pups among pregnant dams) and viability index (% of survival pups on post-natal day 4 after birth) were calculated. Pup individual body weight and sex were recorded on post-natal day (PND) 0 and 4, and these data were reported for each litter. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- Observations of animals during the study period did not reveal any differences in clinical examinations among the treatment and control groups.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No CeO2 NPs-related dead or moribund animals were observed during the study period.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related changes in body weight or weight gain during the study (see Figure 1 in "Attached background material")
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- In male rats of the 300 mg/kg dose group, food consumption during the pre-mating day 1–4 was significantly lower (93% of control) than in vehicle control animals but no alteration were seen afterward. No effect of the treatment was seen in the other treated groups in males and in all groups of treated females as compared to the controls animals (see Table 1 in "Any other information on results incl. tables").vThis finding was considered by the authors to be incidental since it was transient and did not have a dose response.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No overt effect of the treatment was observed as compared to the controls. However, in female rats of the 100 mg/kg dose group, the PT value was significantly higher (1.14-fold over control) than the respective level in the vehicle control animals. Other hematology values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 2 in "Any other information on results incl. tables"). Thus, the significantly increased PT in 100 mg/kg dose-group females was considered to be incidental since it did not have a dose-response.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No overt effect of the treatment was observed as compared to the controls. However, in male rats of the 1000 mg/kg dose group, the GGT value was significantly higher (2.24-fold over control) than the respective level in the vehicle control animals but the other clinical chemistry values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 3 in "Any other information on results incl. tables"). The authors have considered the increase in GGT in 1000 mg/kg dose-group males as incidental since there were no correlated changes in organ weights and histopathological examinations.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- In male rats of the 1000 mg/kg dose group, an increased incidence of tubular basophilia in kidneys (Grade 1) was observed. No such effect were seen in females. No other effect in the examined organs were seen in the treated groups of male and females animals as compared to the controls. The increased incidence of tubular basophilia in the kidneys in 1000 mg/kg dose-group males was considered to be incidental by the autors since it also occurred sporadically in normal animals, did not have an obvious dose response and yield no correlated clinical chemistry changes. In addition, there were no toxicologically significant CeO2 NPs-related changes in other examinations for general systemic effects.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- > FOB :
In functional observations including sensory function tests (tail pinch, approach and touch response, pupillary reflex and acoustic startle response), grip strength and motor activity, there were no treatment-related changes during the study. - Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- No estrus cycle abnormalities was observed in this study.
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related changes in fertility results with precoital time. Mating index, Fertility index and Fecundidity index were all equal to 100 in all groups of males. Mating index, Fertility index and Pregnancy index were also found all equal to 100 whatever the dose level groups in females. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods (see Tables 6 and 7 in "Any other information on results incl. tables").
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No systemic and reproductive effect.
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- In general clinical signs and external examination of F1 pups at necropsy, there were no treatment-related changes among the treatment and control animals.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- No effect of the treatment was seen on the perinatal death and the viability index (see Table 7 in "Any other information on results incl. tables").
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No overt effect of the treatment was seen on pup body weight. An increase in F1 male and female pup covariate-adjusted body weights (up to 1.11-fold over control) during the post-natal period (PND 0 and 4 for males and PND 0 for females) was observed at 1000 mg/kg. (see Table 8 in "Any other information on results incl. tables"). Since there were no concurrent changes in maternal body weight, litter size or gestation length and no concurrent estrus cycle abnormalities and histopathological changes in reproductive organs, therefore, the increased pup body weight was not considered treatment-related.
- Food consumption and compound intake (if feeding study):
- not examined
- 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
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- not examined
- Nipple retention in male pups:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No pup with external abnormalities was found in this study (see Table 7 in "Any other information on results incl. tables").
- Histopathological findings:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No developmental effect observed
- Key result
- Critical effects observed:
- no
- Reproductive effects observed:
- no
- Conclusions:
- In conclusion, under the experimental conditions of this study design, there were no CeO2 NPs related adverse effects in terms of general systemic signs as well as development and reproduction, at doses up to 1000 mg/kg bw/d. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and the NOAEL for developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.
- Executive summary:
In a reproduction/developmental toxicity screening study, the effects of nano CeO2 on the general toxicity and reproductive or developmental toxicity was evaluated following daily oral administration by gavage to Sprague-Dawley rats. The study was performed according to OECD Guideline no 422 and was compliant to GLP. The study was thus considered as a key study of reliablility 1 according to Klimisch score.
Groups of 12 males and 12 females SD rats were treated by gavage with the test substance at dose levels of 0 (controls, vehicle), 100, 300 and 1000 mg/kg/day nano CeO2 in water from 2 weeks before mating, through mating and, for the females, through gestation until lactation day 4, corresponding to 38 days of treatment in males and 41 days of treatment in females. Effect of the treatment on mortality, clinical signs, body weight and body weight gain, food consumption, functional observation battery, hematology and chemical chemistry were evaluated. All animals were sacrificed at the end of the study and gross necropsy and histopathology was performed. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Precoital time and fertility-related data, including mating, fertility, fecundity, pregnancy index and delivery index were calculated. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups were evaluated. Pup mortality, viability index, individual body weight and general clinical signs were examined once daily.
Further, parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected for cerium content analysis using inductively coupled plasma mass spectrometry (ICP-MS).
No unscheduled death or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. No effect of treatment was observed in hematology and clinical biochemistry analyses and in the functional observational battery results. The treatment with the test substance induced no change in organ weight and no gross or histopathological lesion in this study.
No estrus cycle abnormalities was observed in females and no treatment-related changes in fertility results with precoital time was found. No effect of the treatment was observed on the mating index, fertility index, fecundity index and pregnancy index. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods. Pups showed no effect of treatment on survival, clinical signs, body weight and body weight gain. No pup with external abnormalities was found in this study.
Tissue distribution analysis of cerium in parental and pup tissues revealed that nano CeO2 was not detected in almost all of the samples. Only a few samples were slightly above the mean cerium content of blank samples, but it was also observed in vehicle control and there was no correlation in cerium content among the tissues and dose groups.
The authors concluded that, under the experimental conditions of this study, no nano CeO2 related adverse effects on the general systemic signs as well as on the reproductive performance or developmental toxicity was observed at doses up to 1000 mg/kg bw/day. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and the NOAEL for developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Read across based on studies performed with (bulk) zirconium acetate, bulk and nano cerium dioxide, as well as an organometallic form of nano cerium dioxide. The read across justification document is attached to IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause adverse effects on reproduction.
- Remarks:
- Conclusion based on the results of read across studies performed with (bulk) zirconium acetate, bulk and nano cerium dioxide, and an organometallic form of nano cerium dioxide.
- Key result
- Critical effects observed:
- no
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause any adverse effects on reproduction and development.
- Remarks:
- Conclusion based on the results of read across studies performed with (bulk) zirconium acetate, bulk and nano cerium dioxide, and an organometallic form of nano cerium dioxide.
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 24 October 2008 to 13 July 2011
- 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 416 (Two-Generation Reproduction Toxicity Study)
- Deviations:
- yes
- Remarks:
- See details in "Further details on study design". The deviations observed were considered not to have compromised the validity or integrity of the study.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: (P) approximately 6 weeks old for males and approximately 5 weeks old for females; (F1) 3 weeks old (day 22 p.p.).for males and females.
- Weight at study initiation: (P) Males: 204 g to 245 g (mean value: 228 g); Females (P): 142 g to 176 g (mean value: 159 g).
- Fasting period before study: no
- Housing: the F0 males and females and the F1 generation after weaning were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). Towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet.
- Water: free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: 6 days.
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: about 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12hrs dark / 12hrs light
IN-LIFE DATES: from 23 July 2009 (arrival of the animals) to 2 April 2010 (last day of necropsy of F1 animals) - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle and then passed in an ultraturax for at least 5 minutes and until the consistency appeared to be acceptable in order to achieve the nominal concentrations of 37.5, 112.5 and 250 mg/mL. The resulting suspension was left under magnetic stirring until delivery to the animal room and, in the animal room, until treatment of the animals.
The test item dosage forms were prepared daily and were stored in brown flasks at room temperature, protected from light, prior to use.
VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 37.5, 112.5 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg/day
- Lot/batch no.: 017K0127, 058K0070, 049K0043, 128K0040 and MKBC6753 - Details on mating procedure:
- - M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days had elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm). - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- During the pre-study period, the homogeneity and concentration of two dosage forms prepared at concentrations which covered the lowest and highest concentrations of the study (25 and 250 mg/mL) were checked. The analytical method used was ICP-OES.
> Homogeneity and concentration:
Quadruplicate samples were taken from three levels of the container (top, middle and bottom) on the day of preparation. Two samples from each quadruplicate were analyzed and the remaining two samples were stored in case analysis was required. All samples were stored at +4°C and protected from light during shipment. On each occasion, the mean (n = 6) concentration was determined and compared to the nominal value and the coefficient of variation (CV %) was calculated. Acceptance criteria at each time-point: mean concentration = nominal value ± 20%, CV% < 10%.
The results demonstrated the satisfactory homogeneity of the two dosage forms since the differences from nominal concentration were within ± 20% and the % CV was <10%.
> Study chemical analysis - concentration:
The concentration of the test item in the dosage forms was determined in samples of each control and test item dosage form prepared for use in weeks 1, 6, 12, 14 (F0 generation), 17 (F0 and F1 generation), 22, 29 and 33 (F1 generation) of the study.
On each sampling day, duplicate samples were taken from each container. One sample from each duplicate was analyzed and the remaining sample was stored in case analysis was required. On each sampling day, each sample taken was stored at +4°C and protected from light until dispatch for analysis. Acceptance criterion: actual concentration: = nominal value ± 20%.
All dosage forms analyzed were within the ± 20% acceptance criterion. - Duration of treatment / exposure:
- > In the males:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- until sacrifice (after weaning of the pups).
> In the females:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- during pregnancy,
- during lactation until day 21 post-partum (p.p.) inclusive,
- females with no delivery were treated until the day prior to sacrifice. - Frequency of treatment:
- Once a day, 7 days a week
- Details on study schedule:
- - F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation on day 22 post partum.
- Age at mating of the mated animals in the study: between 13 and 14 weeks old. - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 450 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 25 animals/sex/group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: the dose-levels were selected on the basis of the results of a preliminary OECD 422 study using dose-levels of 450 and 1000 mg/kg/day (CIT/Study No. 34759 RSR, April 2010) and a 28-day repeated dose toxicity study (OCDE, 407, CIT Study N° 22314 TSR, January 2002). Neither dose elicited effects on the adult animals although the pups of the group treated at 1000 mg/kg/day had a minimally lower mean body weight gain from post natal days 1 to 5.
- Rationale for animal assignment: computerized stratification procedure, so that the average body weight of each group was similar.
Some deviations from the study plan were observed:
the temperature and relative humidity recorded in the animal room were sometimes outside the target ranges, some F1 animals had no free access to water during a short period of time because of defective water bottles, a total of 9 pups were retained in error from one F1 female (whereas each litter should have been culled to 8 pups on day 4 p.p.), two F0 females (group 1 and group 4) each gave birth to pups after delivery was thought to have been completed, one F0 male (group 2) had a left testis reduced in size which was not sampled at macroscopic examination in error, two F2 pups had scabs on the tail which were not sampled at macroscopic examination in error, statistical analyses performed on primordial follicle and corporea lutea count data were done on mean values per section per animal, instead of total number per animal.
These deviations were considered not to have compromised the validity or integrity of the study. - Positive control:
- not required
- Parental animals: Observations and examinations:
- (F0 and F1 parental generation)
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
> Mortality or signs of morbidity: at least twice a day during the treatment period, and once a day during the acclimation period (F0 only).
> Clinical signs: once a day.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week on all animals until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern).
BODY WEIGHT: Yes
- Time schedule for examinations:
> males: on the first day of treatment (day 1), then once a week until sacrifice.
> females: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1, 4, 7, 14 and 21 p.p.. Females prematurely sacrificed were weighed prior to sacrifice.
FOOD CONSUMPTION: Yes
The quantity of food consumed by each male was recorded once a week, over a 7 day period, from the first day of treatment until sacrifice.
The quantity of food consumed by each female was recorded once a week, over a 7 day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 p.c. intervals) and lactation (days 1-7, 7-14 and 14-21 p.p. intervals) until sacrifice.
During the pairing period, food consumption was not recorded for males or females.
WATER CONSUMPTION: No
OTHER:
- Sexual development (only F1 generation):
> All male animals were observed each day from day 32 of age (i.e. day 11 of F1 generation), until cleavage of the balanopreputial groove (preputial separation) was observed.
> All female animals were observed each day from day 28 of age (i.e. day 7 of F1 generation), until vaginal opening was observed.
The observation period was extended for any animals not positive by the expected end day. Body weight was recorded individually on the positive day.
- Neurobehavioral tests (only F1 generation)
> Auditory function (when the animals were 4 weeks old),
> Pupil constriction (when the animals were 4 weeks old),
> Spontaneous locomotor activity (when the animals were approximately 8 weeks old). - Oestrous cyclicity (parental animals):
- The estrous cycle stage was determined from a fresh vaginal lavage, each morning as follows:
- during the last 3 weeks of the pre-mating period,
- during the mating period, until the females are mated.
(F0 and F1 parental generation) - Sperm parameters (parental animals):
- Parameters examined in F0/F1 male parental generations: testis weight, epididymis weight, sperm count in testes, epididymal sperm motility, epididymal sperm morphology.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: litter size, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical signs, weight gain, physical or behavioural abnormalities (physical development: pinna unfolding (on day 5 p.p.), hair growth (on day 5 p.p.), tooth eruption (on day 13 p.p.), auditory canal opening (on day 17 p.p.,), eye opening (on day 17 p.p.,); and reflex development: surface righting reflex (on day 5 p.p ), cliff avoidance (on day 11 p.p), air-righting reflex (on day 17 p.p).
GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities. - Postmortem examinations (parental animals):
- SACRIFICE
On completion of the treatment period, all surviving males and females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
- F and F1 surviving males: after weaning of the F1 or F2 generation,
- P and F1 surviving females: at the weaning of the litters (on day 22 p.p.),
- P and F1 females which did not deliver: on day 25 p.c. after body weight recording (to check a possible un-noticed delivery),
- P and F1 females with litter dying entirely.
GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all animals including on animals prematurely sacrificed of found dead. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of implantation sites were also recorded for females sacrificed on day 22 p.p..
The numbers of corpora lutea and implantation sites were recorded if possible for females sacrificed on day 25 p.c. due to no delivery. For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively. Furthermore, microscopic examinations were also performed on all animals sacrificed prematurely or found dead, and on all females sacrificed because of no delivery to investigate possible causes.
Testicular staging: a detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with PAS-hematoxylin (groups 1 and 4) in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
A detailed and careful microscopic examination was made of five sections of the right ovary of each F0 and F1 female, with enumeration of the total number of primordial follicles and corpora lutea. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed on day 4 p.p. or on day 22 p.p..
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
The following pups were carefully examined externally for gross external abnormalities:
- pups found dead,
- pups prematurely sacrificed,
- pups culled on day 4 p.p.,
- pups sacrificed on day 22 p.p..
In addition, for the following pups a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. Special attention was paid to the reproductive organs.
- pups showing external abnormalities or clinical signs,
- pups found dead or prematurely sacrificed,
- one randomly selected F1 and F2 pup/sex/litter sacrificed on day 22 p.p..
HISTOPATHOLOGY / ORGAN WEIGTHS
The organs specified in Tissue Procedure Table 2 were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
A microscopic examination was performed on:
- all the tissues listed in the Tissue Procedure Table 2 for one randomly selected F1 pup/sex/litter not selected at weaning and for one randomly selected F2 pup/sex/litter,
- all macroscopic lesions,
- all pups with external abnormalities. - Statistics:
- Body weights, food consumption and reproductive data: mean values were compared by one-way variance analysis and Dunnett test. Percentage values were compared by Fisher exact probability test.
Organ weights: a sequence of statistical tests was used according to PathData software.
Auditory startle reflex: performed using the software SAS Enterprise Guide version 2.05.89.
Numbers of corpora lutea and primary follicles: normality and homogeneity of variances were tested using Kolmogorov Smirnov and Bartlett tests. If normality and homogeneity of variances were demonstrated (p-value>0.5 for both tests), a Student test was implemented. If normality and homogeneity of variances were not demonstrated (p-value<0.05 for one or both of the tests), a Mann-Withney-Wilcoxon test was conducted. - Reproductive indices:
- The following parameters were evaluated:
- Pre-implantation loss,
- Post-implantation loss,
- Mating index,
- Fertility index,
- Gestation index,
- Number of corpora lutea,
- Number of implantations,
- Number of pups delivered - Offspring viability indices:
- The following parameters were evaluated:
- Live birth index,
- Viability index on day 4 post-partum,
- Lactation index on day 21 post-partum,
- Mean litter size,
- Pup sex ratios - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No premature death occurred during the study in F0 male and female rats.
Some females were sacrified during lactation because of clinical signs or dead litter:
- One female at 1000 mg/kg was prematurely sacrificed on lactation day 9 because of clinical signs of piloerection, pallor, hypoactivity, abdominal breathing and half-closed eyes. At microscopic examination, the presence of bacterial colonies in a thrombus of the left cardiac atrium indicated that an infection caused the moribundity of this female.
- Two females at 450 mg/kg were prematurely sacrificed on lactation day 1 because of dead litter. Both females had dead fetuses in the uterine horns at necropsy and both females had necrosis and acute inflammation of the uterus and centrilobular necrosis in the liver at microscopic examination. Since similar lesions were observed in a control female (see below) a relationship to treatment with the test item is considered unlikely.
- One female in the control group was prematurely sacrificed on lactation day 1 because of dead litter. The female had marked centrilobular degeneration/necrosis in the liver, focal and marked necrosis in the uterus.
There were no test item treatment-related clinical signs.
Chromodacryorrhea, reflux at dosing, salivation, nodosities, hairloss, scabs and lesions were all observed at an equal or greater incidence in the controls animals, were considered to be related to the viscosity of the vehicle, corn oil, or are regularly observed in laboratory rats.
One female treated at 450 mg/kg had a mass on the 2nd right mammary gland at the end of the lactation period. This can be observed in lactating rats and given the isolated nature was considered not to be related to treatment with the test item. - Mortality:
- no mortality observed
- Description (incidence):
- No premature death occurred during the study in F0 male and female rats.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- F0 generation (See table 3a):
Mean male body weight gains over the study were similar to those of the controls (between 0% and +5% differences).
The female group treated at 450 mg/kg/day gained statistically significantly more body weight over the 10 week pre-mating period than the controls (+12%). This was due mostly to statistically significantly higher mean body weight gains in weeks 5 and 7. In the absence of any effects at the higher or lower dose-level, this body weight gain difference was considered not to be related to treatment with the test item.
There were no effects of treatment with the test item on mean female body weight gains during the gestation period or during the lactation period at 450 or 1000 mg/kg/day. The mean body weight gain during the lactation period of females treated at 150 mg/kg/day was lower than that of the other groups. This was due to slightly lower body weight gains during the first part of lactation and then slightly greater body weight loss during the second part. Since the groups treated with higher dose-levels were not similarly affected, this lower body weight gain was considered not to be related to treatment with the test item. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Mean male food consumption was statistically significantly higher at 1000 mg/kg/day in weeks 6 and 7 and then from weeks 9 to 16, when compared with the controls. This correlated with greater body weight gains.
The female groups treated at 450 or 1000 mg/kg/day had slightly greater food consumption than the controls at the beginning of the pre-mating period (+9%, p<0.05) and the female group treated at 1000 mg/kg/day again had slightly, but statistically significantly, greater food consumption than the controls at the end of the pre-mating period and during the middle of the gestation period.
There were no effects of treatment with the test item on food consumption during the lactation period. - Food efficiency:
- 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:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Premature deaths:
- 1000 mg/kg/day: In one female sacrificed moribund on lactation day 9, the presence of severe cardiac lesions of septic origin explain the poor clinical status of this animal. This animal had a severe thrombosis of the left cardiac atrium, along with secondary hypertrophy of the right ventricle, and multifocal degeneration/necrosis of the myocardium, the latter correlated with white discoloration at necropsy. Numerous bacterial colonies were found within the thrombus and were most likely secondary to genital infection during delivery. Therefore the cardiac lesions were considered to be incidental and unrelated to the test item administration. In addition, there was a moderate centrilobular necrosis in the liver, correlated with marked lobular pattern at necropsy, and which may also have contributed to the poor clinical condition. Although the pathogenesis is unclear, the presence of a similar lesion in a control female makes the relationship to the test item administration very unlikely. There was a marked, diffuse cortical hypertrophy in the adrenals, correlated with macroscopic enlargement. In the spleen, there was a marked extramedullary hemopoiesis correlated with macroscopic enlargement, and which was considered to be secondary to the ongoing infection and inflammatory lesions. A slight lymphoid atrophy of the spleen and increased porphyrin pigment in the Harderian glands, correlated with black discoloration at necropsy, were considered to be secondary to the stress of the severe cardiac lesions. There were no abnormalities in the genital organs. Placentas were seen in the uterus. The vagina showed marked mucification, as it is awaited during pregnancy and days after delivery.
- 450 mg/kg/day: two females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns.
In one female, moderate acute neutrophilic inflammation and slight multifocal necrosis were found in the uterus (mucosa, lumen and placenta) and correlated with the presence of a dead fetus at necropsy. Slight acute centrilobular necrosis was seen in the liver and likely also contributed to the poor clinical status of this rat. There was a marked lymphoid atrophy of the thymus, which correlated with the gelatinous aspect, and was considered to be secondary to the inflammatory and necrotic lesions in the uterus and liver. In the spleen, there was a marked extramedullary hemopoiesis correlated with macroscopic enlargement, and which was considered to be secondary to the ongoing inflammatory lesions. The second female had similar lesions of necrosis and acute inflammation in the uterus, and of centrilobular necrosis in the liver. In both females, it is unclear whether the uterine findings were primary or secondary to the presence of dead fetuses. Both the uterine and hepatic lesions contributed to their poor clinical status. For both animals, although the pathogenesis of the hepatic necrosis is unclear, the presence of similar necrotic uterine and hepatic lesions in a control female makes the relationship to the test item administration very unlikely. The vagina of both females showed marked mucification, as it is awaited during pregnancy and within days after delivery.
Control: In one female sacrificed on lactation day 1 with a dead litter, marked centrilobular hepatic degeneration/necrosis correlated with white color of the liver. There was a focal, marked necrosis in the uterus in one horn and within the underlying mucosa and placenta, along with multifocal vascular necrosis. Both the hepatic and genital lesions were considered to have contributed to the poor clinical condition of this animal. In the spleen, there was a marked extramedullary hemopoiesis, correlated with macroscopic enlargement, and considered to be secondary to the ongoing inflammatory lesions. There was a diffuse marked lymphoid atrophy of the thymus, correlated with gelatinous aspect at necropsy, and which was secondary to the stress of the lesions. The vagina showed marked mucification, as it is awaited during pregnancy and within days after delivery. There were neutrophils within the vaginal epithelium and lumen, which is not considered to be abnormal after delivery.
Terminal sacrifice:
F0 generation:
> Qualitative evaluation of the genital organs in F0 parents:
There were no significant differences between control and high-dose groups in the incidences and severity of microscopic findings in the genital organs from F0 parents. In particular, at histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
In females which were not pregnant (one at 150 mg/kg, one at 450 mg/kg and three at 1000 mg/kg), there were no microscopic findings in the genital organs attributed to the test item.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
> Quantitative evaluation of the ovaries in F0 females:
There was a slight, statistically significant increase in the mean numbers of corpora lutea in high dose females (12.82 ± 3.047 compared to 10.74 ± 4.631 in control, p<0.01), which correlated with the increase in the mean ovary weights in this group. This variation was not toxicologically significant based on the direction of the change.
There was a slight, not statistically significant, decrease in the mean numbers of primordial follicles in high-dose F0 females. Since such variation was not found in high-dose F1 females, this variation was not considered to be toxicologically significant.
> Microscopic evaluation of the liver and kidneys in F0 parents:
The test item administration at 1000 mg/kg/day induced minimal centrilobular hypertrophy in the liver from 16/25 males, correlated with the increased liver weights. This finding was not adverse. There were no significant changes in the liver from high-dose females.
At this dose-level, the test item administration also induced a minimal increase of the incidence and severity of hyaline droplets in the proximal renal tubules from males, correlated with the increased kidney weights. There were no significant microscopic changes in the kidneys from high-dose females. This change, spontaneously seen in normal mature rats, was reported to be exacerbated by some chemicals but is known to be male rat-specific and therefore has no human relevance. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- It was considered that there were no effects of treatment with the test item on estrous cyclicity in F0 parental generation. (see table 4a)
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on sperm parameters in F0 parental generation. (See table 9a)
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- F0 generation (see table 5a and 6a):
Overall, there were no test item treatment-related effects on mean reproductive parameters and indices.
All males and females mated although the mean number of days taken to mate was greater in all test item-treated groups than for the controls. It is considered however, that a mean of up to 4 days of pairing before mating is within normal range since the rat estrous cycle usually lasts for 4 days and rats mate while in estrus.
There were no more than two non-pregnant females per group which is also considered to be within normal range.
One female treated at 1000 mg/kg/day was pregnant but did not deliver.
The mean duration of gestation was similar in all groups.
The mean number of implantation sites was similar in all groups as were the mean number of pups born and the post-implantation loss. The percentage of male pups was similar in all groups.
The number of dead pups was statistically significantly greater in the groups treated at 450 or 1000 mg/kg/day when compared with the controls. Even when taking into account the litters where all the pups died, pup mortality was still increased at the two highest dose-levels.
At 1000 mg/kg/day, the majority of the dead pups (23/36) were from two litters. One Female had no clinical signs during lactation yet 9/15 of the pups were dead or cannibalized on lactation day 1. the second female had 13 dead pups on lactation day 1 and only one live pup. The pup was still alive on lactation day 9 when the dam was prematurely sacrificed because of poor clinical condition (piloerection, pallor, hypoactivity, abdominal breathing and half-closed eyes). The dam had shown no clinical signs prior to lactation day 9 however microscopic examination revealed the presence of a bacterial infection which is considered to have caused the moribundity of the dam.
At 450 mg/kg/day, the majority of the dead pups were from three litters. The 1st female was pale and had piloerection on lactation day 1 and on the same day all the pups were found dead (7 pups) or cannibalized (5 pups). It is likely that the poor clinical condition of the dam caused lack of nursing or nesting behavior resulting in death of the pups. The 2nd female also had an entire dead litter on lactation day 1 but had no clinical signs. The 3rd female lost 10/14 pups on lactation day 1 (9 were found dead and 1 was cannibalized) but had no clinical signs. The remaining four pups survived until weaning. The 2nd and 3rd females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns, along with necrotic uterine and hepatic lesions which were most probably the main cause of their clinical signs and/or secondary lack of nursing. In view of their low incidence and presence of similar lesions in a control female, the relationship to the test item was considered to be unlikely.
One control female also had a dead litter on lactation day 1 (13 found dead pups and 1 cannibalized pup) and had piloerection and pallor. The clinical signs could indicate poor maternal condition after delivery. At microscopic examination, there were necrotic hepatic and uterine lesions which were considered to have contributed to its poor clinical condition.
This distribution of pup mortality (the majority of the dead pups being from a small number of litters) is more indicative of poor maternal care rather than a direct effect of the test item on pup mortality, which would probably cause more widespread pup death rather than being concentrated in some litters. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- (parental F0)
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no significant effect observed in the reproductive performance and systemic toxicity
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- 1000 mg/kg/day: One female was found dead on day 48. Clinical signs of pallor, hypoactivity, piloerection, chromorhinorrhea and half-closed eyes had been observed from day 46 or day 47. Death was related to marked suppurative pyelonephritis and cystitis which were considered to be incidental and unrelated to the test item administration.
Two females were sacrificed on gestation day 23 because of difficulties to deliver evidenced by clinical signs of pallor and piloerection: one female had a fetus in the vagina but had not delivered any pups, the other female had delivered eight pups (seven live and one dead) but parturition was not complete. At microscopic examination, there were necrotic uterine lesions along with dead fetuses. A relationship to the test item administration was considered to be unlikely in view of their low incidence and presence of similar uterine lesions in a control female.
- 450 mg/kg/day: One female was found dead on day 10 of dosing. No clinical signs had been observed before death. Death was related to gavage pneumonia and was therefore unrelated to the test item.
- 150 mg/kg/day: One male was prematurely sacrificed on day 126 of treatment because of clinical signs of piloerection, round back, bent head, hypoactivity, loud breathing, half-closed eyes, swollen neck region and chromorhinorrhea. At necropsy, this male had an esophageal pouch which correlated with marked acute inflammation microscopically. This finding explained the clinical signs observed and was secondary to a dosing error.
- Control: One control female was prematurely sacrificed on gestation day 24 because of difficulties to deliver. The female had delivered 13 pups (10 live and 3 dead) but was pale and it was considered that delivery was not complete. At microscopic examination, there was a marked suppurative inflammation of the uterus. Another female was prematurely sacrificed on lactation day 7 because of dead litter: there were no macroscopic or microscopic findings.
There were no test item treatment-related clinical signs. Signs of lesions, hairloss and nodosities were also observed in control animals and are often seen in laboratory rats. One male treated at 1000 mg/kg/day had a mass on the left forelimb which increased in size from 2 x 1 cm on day 113 to 4 x 4 cm on the day of sacrifice (day 130). At microscopic examination, this mass correlated with a subcutaneous sarcoma which was considered to be incidental. Such malignant tumors have been reported in the literature in rats of this age (Son and Gopinath, 2004). - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- see clinical signs
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on mean male body weight or body weight gain during the 10-week premating treatment period. By the end of the study, the test item treated males had gained more weight than the controls and mean body weights were 3% to 4% higher.
Females treated at 150 mg/kg/day had greater mean body weight gains than the controls throughout the pre-mating and gestation periods, and had statistically significantly higher mean body weights throughout gestation and most of lactation (+6% to +9%, p<0.05, p<0.01 or p<0.001) although the overall mean body weight gain did not achieve statistical significance. The group treated at 1000 mg/kg/day had statistically significantly higher mean body weight gains during the first two weeks of gestation which contributed to a non-statistically significantly higher overall body weight gain.
Mean body weight gains over lactation were similar to that of the controls.
While treatment-related, all these changes were considered of not toxicological significance.
(See table 3b) - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Mean food consumption of the males treated at 1000 mg/kg/day was statistically significantly higher than that of the controls from week 7 until the end of the study. The mean food consumption of the group treated at 450 mg/kg/day was also statistically significantly higher from week 12.
The female group treated at 1000 mg/kg/day showed a similar effect, having statistically significantly higher food consumption from week 9 until mid-gestation, when compared with the controls.
In all treated groups mean food consumption of the females from treated groups was slightly, but non-statistically significantly, higher than that of the controls during lactation. - Food efficiency:
- 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:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The test item administration was associated with statistically significant increases in the mean kidney and liver weights in F1 male parents. However, these changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
There were increases of the mean absolute (statistically significant) and relative weights of seminal vesicles in mid-dose and low-dose males compared with controls. In the absence of similar variations in high-dose males and since there were no macroscopic and microscopic correlates, this finding was considered to be incidental and unrelated to the test item administration.
The test item did not induce any significant change in all treated groups of F1 females as compared to controls.
The test item administration in F1 parents did not induce any changes in organ weights in their pups.
(see table 8) - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Premature deaths:
- 1000 mg/kg/day: one found dead female (day 48) had many white discolored foci in the kidneys, unilaterally dilated renal pelvis, dilated urinary bladder with red liquid content, and enlarged adrenals. In two females sacrificed because of difficulties to deliver (day 23), there were dead fetuses and black content within both uterine horns and red content in the vagina ; in one female, all 17 fetuses remaining in the uterine horns were found dead, whereas in the other female, four fetuses were found dead and one fetus was still alive.
- 450 mg/kg/day: In one female, found dead on day 10, the lungs were enlarged.
- 150 mg/kg/day: a male sacrificed moribund on day 126 had a large pouch (2.5 cm long) around the esophagus with white thick content.
- Control: In one control female sacrificed because of difficulties to deliver (day 24), there was still one fetus within the uterus and two placentas in the vagina. The spleen was enlarged. In another female sacrificed on lactation day 7 because of dead litter, there were no macroscopic findings.
Terminal sacrifice:
The few macroscopic findings noted at the end of the treatment period in F1 parents, as well as in their pups were of those commonly recorded in the Sprague-Dawley rat, and none were considered to be related to the test item administration. Among them was a large white mass of the forelimb of high-dose parent male S24963, which correlated microscopically with a subcutaneous sarcoma. Such malignant tumors have been reported in the literature in rats of this age (Son and Gopinath, 2004). - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Premature deaths:
- 1000 mg/kg/day: In found dead female (day 48), there was a marked suppurative inflammation of the kidneys (pyelonephritis) correlated with the macroscopic white foci and pelvic dilation, urinary bladder (cystitis correlated with dilation and red content) and tissue adjacent to the vagina. Numerous bacterial colonies were seen in the kidneys. These suppurative lesions were the cause of death of this female. There was a moderate diffuse hypertrophy of the cortex correlated with enlargement, and which was considered to be secondary of the stress of the multiple suppurative lesions in this animal. Pyelonephritis and cystitis are common findings in female rats, and in this case were considered to be incidental and unrelated to the test item administration. In two females sacrificed because of difficulties to deliver (day 23), most fetuses were dead. In the uterus, there was slight acute focal necrosis on the endometrial surface at the level of one placenta in both females, along with slight multifocal hemorrhage in one female and minimal multifocal neutrophilic inflammation in the other female. It is unclear whether the uterine lesions were primary or secondary to the presence of dead fetuses. All these findings likely contributed to the clinical signs of the females. A relationship to the test item administration of the fetal deaths was considered to be unlikely in view of their low incidence and presence of similar uterine lesions in a control F0 female.
- 450 mg/kg/day: In the female found dead on day 10, there was a diffuse gavage pneumonia characterized by the presence of yellow material interpreted as compound within the alveoli. This pneumonia, correlated with the macroscopic enlargement, was considered to be the cause of death.
- 150 mg/kg/day: the esophageal pouch of the male sacrificed moribund on day 126 correlated with marked acute inflammation microscopically. This finding explained the clinical signs observed and was secondary to a dosing error.
Control: in the control female sacrificed because of difficulties to deliver (day 24), there was a marked acute neutrophilic inflammation of the uterus related to the presence of bacterial colonies, associated with multiple necrotic areas in the mucosa/placentas and focal thrombosis. It is difficult to determine whether the septic inflammation was the cause or the consequence of the difficulties to deliver. There was marked hemopoiesis in the spleen which correlated with splenic enlargement, and was considered to be compensatory to the ongoing inflammatory lesions. In the control female sacrificed on lactation day 7 because of dead litter, there were no significant microscopic findings in the genital organs.
Terminal sacrifice:
> Qualitative evaluation of the genital organs in F1 parents:
There were no significant differences between control and high-dose groups in the incidences and severity of microscopic findings in the genital organs from F1 parents. . In particular, at histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
In a female at 150 mg/kg/day which was not pregnant and was sacrificed on day 99, there was evidence of a persistent estrus, characterized by multiple follicular cysts and absence of corpora lutea in the ovaries, tall columnar endometrial epithelium and squamous metaplasia in the uterus, and epithelial hyperplasia and cornification in the vagina (Westwood, 2008). This isolated condition, not observed in high-dose females, was considered to be incidental and unrelated to the test item administration.
In females which were not pregnant (one in control, three at 150 mg/kg, two at 450 mg/kg and two at 1000 mg/kg), there were no microscopic findings in the genital organs attributed to the test item.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
> Quantitative evaluation of the ovaries in F1 females:
There were no significant differences in the mean number of primordial follicles between control and high-dose F1 females. In the right ovary of one high-dose female, there were no primordial follicles and there was a small number of corpora lutea (n=9), which correlated with a reduced size macroscopically. Since the left ovary of this female was not affected, this unilateral change was considered to be incidental and unrelated to the test item administration.
There was a slight, not statistically significant increase in the mean number of corpora lutea in high-dose F1 females compared with controls. This variation was not considered to be toxicologically significant based on the direction of the change.
> Microscopic evaluation of the liver and kidneys in F1 parents:
The test item administration at 1000 mg/kg/day induced minimal centrilobular hypertrophy in the liver from 10/25 males, correlated with the increased liver weights. This finding was not adverse. There were no significant changes in the liver from high-dose females.
There were no significant differences in the incidences of hyaline droplets in the kidneys from high-dose males compared with controls. There were no significant findings in the kidneys from high-dose females.
All microscopic findings noted in treated F0/F1 parent animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat. - Histopathological findings: neoplastic:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- It was considered that there were no effects of treatment with the test item on estrous cyclicity in F1 parental generation. (see table 4b)
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on sperm parameters in F1 parental generation. (See table 9b)
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- See tables 5b and 6b.
There were no treatment-related effect on mean reproductive parameters and indices.
Two males treated at 150 mg/kg/day did not mate but all other animals mated and the mean number of days of pairing before mating was considered not to have been affected by treatment with the test item. There were four non-pregnant females at 150 mg/kg/day compared with one in the control group. There were no microscopic findings in the genital organs of these females or the males which were attributed to the test item. One female from the control group and two females from the high-dose group were sacrificed mid-parturition because of poor clinical condition.
The mean duration of gestation was identical in all groups.
There were no statistically significant differences between control and treated groups regarding mean numbers of implantations, delivered pups and post-implantation loss.
Pup mortality was not increased in the test item-treated groups and there was no effect of treatment on the mean percentage of male pups.
Generally, the pups which were later cannibalized or found dead did not have clinical signs. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no significant effect observed in the reproductive performance and systemic toxicity
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were few clinical signs in surviving pups during lactation, scabs and small wounds are regularly observed, and only one surviving pup from the dam S25228, given 450 mg/kg/day, had dehydration and coldness on days 6 to 8 p.p..
Scabs and hematomas are regularly observed in young rats and incidences of coldness were observed in control pups as well as in those from the groups treated at 450 or 1000 mg/kg/day. - Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- The number of dead pups was statistically significantly greater in the groups treated at 450 or 1000 mg/kg/day when compared with the controls. Even when taking into account the litters where all the pups died, pup mortality was still increased at the two highest dose-levels.
At 1000 mg/kg/day, the majority of the dead pups (23/36) were from two litters. One Female had no clinical signs during lactation yet 9/15 of the pups were dead or cannibalized on lactation day 1. The second female had 13 dead pups on lactation day 1 and only one live pup. The pup was still alive on lactation day 9 when the dam was prematurely sacrificed because of poor clinical condition (piloerection, pallor, hypoactivity, abdominal breathing and half-closed eyes). The dam had shown no clinical signs prior to lactation day 9 however microscopic examination revealed the presence of a bacterial infection which is considered to have caused the moribundity of the dam.
At 450 mg/kg/day, the majority of the dead pups were from three litters. The 1st female was pale and had piloerection on lactation day 1 and on the same day all the pups were found dead (7 pups) or cannibalized (5 pups). It is likely that the poor clinical condition of the dam caused lack of nursing or nesting behavior resulting in death of the pups. The 2nd female also had an entire dead litter on lactation day 1 but had no clinical signs. The 3rd female lost 10/14 pups on lactation day 1 (9 were found dead and 1 was cannibalized) but had no clinical signs. The remaining four pups survived until weaning. The 2nd and 3rd females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns, along with necrotic uterine and hepatic lesions which were most probably the main cause of their clinical signs and/or secondary lack of nursing. In view of their low incidence and presence of similar lesions in a control female, the relationship to the test item was considered to be unlikely.
One control female also had a dead litter on lactation day 1 (13 found dead pups and 1 cannibalized pup) and had piloerection and pallor. The clinical signs could indicate poor maternal condition after delivery. At microscopic examination, there were necrotic hepatic and uterine lesions which were considered to have contributed to its poor clinical condition.
This distribution of pup mortality (the majority of the dead pups being from a small number of litters) is more indicative of poor maternal care rather than a direct effect of the test item on pup mortality, which would probably cause more widespread pup death rather than being concentrated in some litters. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no effects of treatment with the test item on mean pup body weight or body weight gains at any dose-level.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- no effects observed
- Description (incidence and severity):
- In F1 generation, there were no treatment-related effects on balanopreputial separation at any dose-level.
There were no test item treatment-related effects on vaginal opening at any dose-level. The later mean age of vaginal opening at 150 and 450 mg/kg/day was due to one female per group which was considered not to have achieved complete opening before mating. - Anogenital distance (AGD):
- not examined
- Nipple retention in male pups:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- The test item administration in F0 parents did not induce any changes in organ weights in F1 pups.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- On external examination of F1 pups during lactation period, one female pup from one litter had generalized palor, scab and nodositie on abdomen on day 4 p.p. and, another female pup from another litter had a knicked tail on day 22 p.p. in the 1000 mg/kg/day group, one female pup had scab on head on day 4 p.p. in the 450 mg/kg/day group and one female pup had scab on abdomen on day 4 p.p. in the 150 mg/kg/day group. All these findings were isolated and a relationship to the treatment was considered unlikely.
- Histopathological findings:
- not examined
- Description (incidence and severity):
- As no macroscopic lesion was observed in F1 pups at external examination, no microscopic examination was performed.
- Other effects:
- no effects observed
- Description (incidence and severity):
- > Physical and reflex development during lactation (F1 pups):
All pups were positive for pinna unfolding and hair growth on post-natal day 5, tooth eruption on day 13 p.p. and auditory canal opening on day 17 p.p..
Not all pups were positive for eye opening on day 17 p.p., however the incidence was identical to the control group (one pup per group in groups 1 and 4).
The number of pups passing the surface righting and air righting tests was similar to or greater than in the control group at all dose-levels.
The number of pups failing the cliff avoidance test on day 11 p.p. was slightly higher at 450 and 1000 mg/kg/day than in the control group. In the absence of any effects on any of the other physical or reflex development tests, this difference was considered to be incidental.
> Auditory function (F1 pups) :
There was no treatment-related effect on auditory function.
All animals had a positive response to the auditory startle test. The latency and amplitude of the response was similar in all groups therefore it was concluded that no group showed impairment of hearing.
> Pupil constriction and locomotor activity (F1 pups)
There was no treatment-related effect on pupil constriction and locomotor activity.
All groups had similar numbers of horizontal and rearing movements during the 1-hour period as the controls.
All animals were positive for pupil constriction reflex at 4 weeks of age. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Remarks:
- (offspring)
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no significant effect observed on the development of pups
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no test item treatment-related clinical signs.
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- Pup mortality was not increased in the test item-treated groups;
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- the pups from the group treated at 150 mg/kg/day had a statistically significantly greater mean body weight gain between days 7 and 14 p.p. which resulted in higher mean body weights of both male and female pups at the end of lactation. There were no effects at 450 or 1000 mg/kg/day.
In the absence of any dose-related effect a relationship to the treatment was considered unlikely.
(see table 11) - Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- not examined
- Nipple retention in male pups:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- The test item administration in F1 parents did not induce any changes in organ weights in F2 pups.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related findings at external examination of the pups.
- Histopathological findings:
- not examined
- Description (incidence and severity):
- As no macroscopic lesion was observed in F2 pups at external examination, no microscopic examination was performed.
- Other effects:
- no effects observed
- Description (incidence and severity):
- > Physical and reflex development (F2):
There were no treatment-related effects.
All pups were positive for pinna unfolding and hair growth on day 5 p.p. and tooth eruption on day 13 p.p..
Not all pups were positive for eye opening or auditory canal opening on day 17 p.p., however the incidence was nearly identical to the control group.
The number of pups passing the surface righting, cliff avoidance and air righting tests was similar to or greater than in the control group at all dose-levels. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F2
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no significant effect observed on the development of pups
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- The No Observed Adverse Effect Level for the F0 and F1 generations was considered to be >= 1000 mg/kg bw/day.
The test substance did not cause delayed or impaired development in the F1 or F2 generations following treatment of the parents. Therefore, the No Observed Effect Level (NOEL) for peri- and post-natal development of F1 and F2 generations was considered to be >= 1000 mg/kg/day. - Executive summary:
In a 2-generation reproduction study scored as validity 1 according to Klimisch criteria (CIT report No. 34760 RSR, OECD guideline 416, GLP), four groups of 25 male and 25 female Sprague-Dawley rats received the test item daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. The test item was administered as a suspension in corn oil, by oral gavage, at dose-levels of 0 (control), 150, 450 or 1000 mg/kg/day. A constant dosage volume of 4 mL/kg/day was used.
The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once a week. Body weight and food consumption were recorded weekly. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening and, surface righting, cliff avoidance and air righting reflexes).
After weaning of the pups, the males and females of the F0 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs of the control and high-dose group animals and on all macroscopic lesions.
F1 generation
After weaning (day 22post-partum) of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of four groups of 25 male and 25 female rats. Three groups received the test item and the fourth group (control) received the vehicle only (corn oil) daily for 10 weeks prior to mating and, during mating, gestation and lactation until weaning of the pups. The test item was administered as a suspension in corn oil, by oral gavage, at dose-levels of 150, 450 or 1000 mg/kg/day under a constant dosage‑volume of 4 mL/kg/day.
The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once a week. Body weight and food consumption were recorded weekly. Each animal was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight was recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, andweeks of age, spontaneous locomotor activity was measured using an automated infra-red sensor equipment. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 16 days.
The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development was assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening and, surface righting, cliff avoidance and air righting reflexes).
At weaning of the pups, the males and females of the F1 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs of the control and high-dose groups and on all macroscopic lesions.
In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.
Results
F0 generation
There were no found dead animals. A total of four females (one at 1000 mg/kg/day, two at 450 mg/kg/day and one in controls) were prematurely sacrificed during lactation, and microscopic examination findings excluded a relationship to the test item.
No test item treatment-related clinical signs were observed and there were no treatment-related effects on body weight or body weight gain. There was a statistically significant increase in mean food consumption in males and females treated at 1000 mg/kg/day during the pre-mating period and mid-gestation and in females treated at 450 mg/kg/day at the beginning of the pre-mating period.
There were no effects on estrous cyclicity, mating or fertility parameters at any dose-level, however pup mortality was statistically significantly higher at 450 and 1000 mg/kg/day. One female at 1000 mg/kg/day and one female at 450 mg/kg/day had clinical signs which could indicate poor maternal nesting or nursing behavior. Few of the dead or cannibalized pups from the other litters had clinical signs. Since the majority of the found dead pups were concentrated in a few litters, it is considered unlikely that their deaths were related directly to test item treatment and that it is more probably related to poor maternal nesting/nursing behavior. The presence of one dead litter in the control group suggests that the dead litters were incidental to treatment with the test item.
No effects on spermatogenesis were detected after analysis of epididymidal sperm motility and morphology and count and, after analyis of testicular sperm count. At histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle. At all dose-levels, there were a few organ weight changes which were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
There were no test item‑related microscopic findings in the genital organs of F0 parents.
There was a statistically significant increase in the mean ovary weight in F0 females treated at 1000 mg/kg/day, correlating with an increase in the mean number ofcorpora lutea. This finding was not considered to be toxicologically significant based on the direction of the change. In this group, mean number of primordial follicles was slightly decreased. Since such variation was not found F1 females, this variation was not considered to be of toxicological significance.
F1 generation
The F1 generation showed no effects of treatment while pups; there were no test item treatment‑related clinical signs, no effects on body weight and no differences in physical or reflex development when compared with the controls.There were no treatment-related findings at external examination of the F1 pups.
There were no test item treatment-related mortalities.
As for the F0 generation, the males treated at 450 or 1000 mg/kg/day and the females treated at 1000 mg/kg/day had statistically significantly increased food consumption which correlated with a tendency towards a non statistically significant increase in body weight gains. The females treated at 150 or 450 mg/kg/day also had slightly increased food consumption during lactation.
There were no effects on estrous cyclicity or mating.
Pup mortality was not higher in test item-treated groups than in the controls.
No effects on spermatogenesis were detected after analysis of epididymidal sperm motility, morphology or count or testicular sperm count. At histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
The test item administration at all dose-levels induced statistically significant dose-related increases in mean kidney and liver weights in F1 males. On microscopic examiniation, the increase in mean liver weight was correlated with minimal centrilobular hypertrophy in males at 1000 mg/kg/day. There were no microscopic correlates in the kidneys. There were no effects on organ weights and no relevant microscopic findings in the liver and kidneys in F1 females.
There were no test item-related macroscopic findings in F1 parents. There were no test item‑related microscopic findings in the genital organs from F1 parents.
There were increases in the mean number ofcorpora luteain high-dose F1 females. This finding was not considered to be toxicologically significant based on the direction of the change. There were no significant differences in the mean number of primordial follicles between control and high‑dose F1 females.
F2 generation
There were no test item treatment-related clinical signs and no effects on physical or reflex development.
The pups from the groups treated at 150 mg/kg/day had statistically significantly higher mean body weight gains mid-lactation but there were no effects at 450 or 1000 mg/kg/day. Therefore a relationship to the treatment with the test-item was considered unlikely.
There were no test item-related changes in organ weights or macroscopic findings in the pups.
The No Observed Adverse Effect Level for reproductive performance and systemic toxicity of F0 and F1 generations was therefore considered to be >= 1000 mg/kg/day.
The test substance did not cause delayed or impaired development in the F1 or F2 generations following treatment of the parents.
Therefore, the No Observed Effect Level (NOEL) for peri- and post-natal development of F1 and F2 generations was considered to be >= 1000 mg/kg/day.No classification for reproductive toxicity is warranted based on the absence of relevant effects in this study, according to the criteria of Annex VI Directive 67/548/EEC or UN/EU GHS.
This study is classified as acceptable. It satisfies the OECD 416 guideline requirements on two-generation reproduction toxicity testing.
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- Read across based on a study performed with an organometallic form of nano cerium dioxide. The read across justification document is attached to IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause adverse effects on reproduction.
- Remarks:
- Conclusion based on the results of a read across study performed with an organometallic form of nano cerium dioxide.
- Key result
- Critical effects observed:
- no
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause adverse effects on reproduction.
- Remarks:
- Conclusion based on the results of a read across study performed with an organometallic form of nano cerium dioxide.
- Key result
- Critical effects observed:
- no
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause any adverse effects on reproduction and development.
- Remarks:
- Conclusion based on the results of a read across study performed with an organometallic form of nano cerium dioxide.
- Key result
- Critical effects observed:
- no
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause adverse effects on reproduction and development.
- Remarks:
- Conclusion based on the results of a read across study performed with an organometallic form of nano cerium dioxide.
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
Referenceopen allclose all
- No mortality occurred in the study.
- No clinical findings of toxicological significance were observed. Hair loss was occasionally recorded throughout the study including control animals. One female of the mid-dose group had salivation on Day 20 post coitum. One high dose female, that did not give birth, showed prolapse of the uterus on Day 27 post coitum. Another high dose female had rales during pairing.
BODY WEIGHT AND WEIGHT GAIN
- Body weight and body weight gain did not show relevant differences between groups. In particular, body weight gain was in some occasions higher in treated groups compared to the control group.
HAEMATOLOGY
- No changes of toxicological relevance were recorded.
- A statistically significant decrease of lymphocytes recorded in some females dosed with 300 mg/kg bw/day (42%) was not dose-related and, therefore, is considered incidental.
- No changes were observed in the coagulation test.
CLINICAL CHEMISTRY
- A number of males in the high dose group showed a slight decrease of protein and globulin (approximately 10%). Due to the low severity, these changes were considered of no toxicological importance.
- In addition, one animal showed high triglycerides (6.2 fold compared with controls). Due to the low incidence, this finding cannot be conclusively attributed to treatment; however, it also cannot be ruled out that it was related to treatment.
NEUROBEHAVIOUR
- Motor activity recorded at the end of treatment did not show significant differences between control and treated groups.
ORGAN WEIGHTS
- A slight significant reduction of epididymides weight occurred in the high-dose group; however, this reduction was found to be related to the higher terminal body weight in the high-dose group compared to controls. In addition, this change was minimal and no histological associated findings were found. Therefore, it was considered of no toxicological relevance.
TERMINAL BODY WEIGHT AND ORGAN WEIGHTS
Body weight at term and organ weights did not show differences of toxicological relevance.
HISTOPATHOLOGY: NON-NEOPLASTIC
- Minimal, focal vacuolation of squamous epithelium (limiting ridge) of the non-glandular region of the stomach was observed was observed in the high and mid-dose males with an increased incidence in the high dose males and similar severity levels across treatment groups. However, as this gastric change was noted only in males, in a specific zone of the forestomach (limiting ridge) with focal and minimal severity and since humans do not have forestomach (squamous epithelium), such change could be considered toxicologically irrelevant.
- The remaining lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.
OTHER FINDINGS:
Spermatogenic cycle:
- A detailed qualitative examination of the testes was performed in control and high dose groups. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Oestrous cycle, reproductive parameters, pairing combination and mating performance:
No differences were found in the number of oestrous cycle, pre-coital intervals, copulatory and fertility indices between treated and control groups.
Implantation, pre-birth loss data and gestation length of females:
No significant differences were observed in the number of implantations, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups.
- Small pups were generally observed in all groups including the control group.
- Cold to touch and/or apparently no food intake were also occasionally recorded in all groups.
- One pup of low dose group showed absence of tail. This abnormality was considered incidental.
BODY WEIGHT (OFFSPRING)
- Mean pup weights were found comparable between groups at birth and on Day 4 post partum. Sex ratio did not differ between groups.
GROSS PATHOLOGY (OFFSPRING)
- The majority of decedent pups had autolysed organs in the abdominal cavity at necropsy.
- No abnormalities were found in pups sacrificed on Day 4 post partum with the exception of the low dose pup with the absence of tail. One pup of the mid-dose group showed no milk in stomach.
LITTER DATA AND SEX RATIOS:
Litter data including mean litter and pup weights were comparable between groups. No differences were found in sex ratio.
Formulation
The overall results of the test formulation analyses were within the limits of acceptance for concentration (15% of the theoretical concentration).
Parameters
| Reproductive parameters | Group 1 (0 mg/kg bw/day) | Group 2 (100 mg/kg bw/day) | Group 3 (300 mg/kg bw/day) | Group 4 (1000 mg/kg bw/day) |
| Pre-coital interval (days) | 3.3 | 3.0 | 2.9 | 2.9 |
| Copulatory Index (%) | 100.0 | 100.0 | 100.0 | 100.0 |
| Fertility index (%) | 90.0 | 100.0 | 100.0 | 100.0 |
| Gestation length (days) | 21.88 | 22.10 | 22.0 | 22.11 |
| No. of pregnant females | 9/10 | 10/10 | 10/10 | 10/10 |
| No. of non pregnant females | 1/10 | 0/10 | 0/10 | 0/10 |
| No. of females with litter at birth | 9/10 | 10/10 | 10/10 | 9/10 |
| No. of females with litter on Day 4 post partum | 9/10 | 10/10 | 10/10 | 9/10 |
| No. of pregnant females w/o litter | 0/10 | 0/10 | 0/10 | 1/10 |
| Litter data | Group 1 (0 mg/kg bw/day) | Group 2 (100 mg/kg bw/day) | Group 3 (300 mg/kg bw/day) | Group 4 (1000 mg/kg bw/day) |
| Total litter size at birth | 13.67 | 15.40 | 15.60 | 15.33 |
| Live litter size at birth | 13.67 | 15.20 | 15.50 | 15.22 |
| Live litter size at Day 4 post partum | 12.78 | 14.80 | 15.40 | 14.33 |
| Sex ratio at birth (% males) | 57.76 | 58.54 | 49.05 | 55.53 |
| Sex ratio on Day 4 post partum (% males) | 58.01 | 59.62 | 49.60 | 59.98 |
Sex ratio: the percentage of male pups was similar between all groups.
Table 2: summary table of reproductive data
Dose level (mg/kg/d) |
|
0 |
150 |
450 |
1000 |
Paired males + females |
n |
10 + 10 |
10 + 10 |
10 + 10 |
10 + 10 |
Pairs mated |
n |
10 |
10 |
10 |
10 |
Mating index |
% |
100.0 |
100.0 |
100.0 |
100.0 |
Mean number of days of pairing before mating |
n |
2.7 |
3.6 |
3.4 |
2.1 |
Pregnant female partners |
n |
10 |
8 |
9 |
7 |
Fertility index |
% |
100.0 |
80.0 |
90.0 |
70.0 |
Females with live concepti |
n |
10 |
8 |
9 |
7 |
Gestation index |
% |
100.0 |
100.0 |
100.0 |
100.0 |
Table 3: summary table of delivery data
Dose level (mg/kg/d) |
0 |
150 |
450 |
1000 |
Number of pregnant females surviving delivery Mean duration of gestation (days) Mean number of corpora lutea Mean number of implantations Mean pre-implantation loss (%) Mean number of pups delivered Mean post-implantation loss (%) |
10 21.9 19.4 18.2 5.8 14.7 18.7 |
8 21.9 18.6 16.1 12.9 14.1 12.1 |
9 21.9 19.9 17.1 12.4 15.4 9.4 |
7 21.9 19.1 16.1 15.2 14.3 12.7 |
Table 4: summary table of reproductive and litter data
Dose level (mg/kg/d) |
|
0 |
150 |
450 |
1000 |
Females on Study |
N |
10 |
10 |
10 |
10 |
Females Mated Mating Index |
N % |
10 f 100.0 |
10 100.0 |
10 100.0 |
10 100.0 |
Females Pregnant Female Fertility Index |
N % |
10 f 100.0 |
8 80.0 |
9 90.0 |
7 70.0 |
Females with Liveborn Gestation Index |
N % |
10 f 100.0 |
8 100.0 |
9 100.0 |
7 100.0 |
Females Surviving Delivery
Duration of gestation
With stillborn pups
With all stillborn
With entire liveborn litter dying and/or missing, cannibalized, culled Days 0-4
Days 0-5
|
N
Mean S.D.
N %
N %
N %
N % |
10 f
21.9 d 0.3
0 f 0.0
0 f 0.0
0 f 0.0
0 f 0.0 |
8
21.9 0.4
0 0.0
0 0.0
0 0.0
0 0.0 |
9
21.9 0.3
0 0.0
0 0.0
0 0.0
0 0.0 |
7
21.9 0.4
0 0.0
0 0.0
0 0.0
0 0.0 |
Litters with liveborn pups |
N |
10 |
8 |
9 |
7 |
Pups Delivered (total)
Liveborn Live birth index
Stillborn
Culled (total) Cannibalized Missing Died
Liveborn, not culled prior to day 5 |
N Mean S.D.
N %
N %
N N N N
N |
147 14.7 d 1.6
147 f 100.0
0 f 0.0
0 1 0 2
147 |
113 14.1 2.3
113 100.0
0 0.0
0 1 0 1
113 |
139 15.4 2.2
139 100.0
0 0.0
0 0 0 2
139 |
100 14.3 4.0
100 100.0
0 0.0
0 0 0 2
100 |
Pups dying, missing, and/or cannibalized Day 0
Days 1-4
Days 5-7
|
N %
N %
N % |
0 f 0.0
3 f 2.0
0 f 0.0 |
0 0.0
2 1.8
0 0.0 |
0 0.0
2 1.4
0 0.0 |
0 0.0
2 2.0
0 0.0 |
Pups surviving 4 days Viability index |
N % |
144 f 98.0 |
111 98.2 |
137 98.6 |
98 98.0 |
Pups surviving 5 days Lactation index |
N % |
144 f 100.0 |
111 100.0 |
137 100.0 |
98 100.0 |
Implantation sites per litter |
N Mean S.D. |
182 18.2 d 2.1 |
129 16.1 2.5 |
154 17.1 2.1 |
113 16.1 2.5 |
Corpora lutea |
Total Mean S.D. |
194 19.4 d 2.3 |
149 18.6 2.2 |
179 19.9 3.9 |
134 19.1 2.0 |
Preimplantation loss |
Mean % S.D. |
5.8 d 8.2 |
12.9 13.4 |
12.4 11.6 |
15.2 12.7 |
Live pups / litter Day 1
Day 4
Day 5
|
Mean S.D.
Mean S.D.
Mean S.D. |
14.5 d 1.6
14.4 d 1.6
14.4 d 1.6 |
14.0 2.2
13.9 2.3
13.9 2.3 |
15.3 2.1
15.2 1.9
15.2 1.9 |
14.1 4.0
14.0 4.3
14.0 4.3 |
Pup weight / Litter (grams) Day 1
Day 5
|
Mean S.D.
Mean S.D. |
7.4 d 0.6
11.3 d 0.7 |
7.7 0.6
12.1 1.5 |
7.6 0.7
11.7 1.5 |
7.5 0.5
12.2 1.9 |
Sex ratio – male pups: total pups Day 0
Day 5
|
N %
N % |
73 f 49.7
71 f 49.3 |
51 45.1
51 45.9 |
76 54.7
74 54.0 |
50 50.0
49 50.0 |
Statistical key: d = ANOVA + Dunnett-test; f = Fishers exact test; N = number of animals
Summary table of mating and fertility data
Dose level (mg/kg/d) |
0 |
450 |
1000 |
Number of animals paired (M + F) |
10 + 10 |
10 + 10 |
10 + 10 |
Number of males mated |
9 |
10 |
10 |
Number of females mated |
10 |
10 |
10 |
Mean number of days taken to mate |
5.2 |
5.6 |
2.3 |
Number of pregnant females |
10 |
10 |
8 |
Summary table of delivery data
Dose level (mg/kg/day) |
0 |
450 |
1000 |
Number of pregnant females surviving delivery Mean duration of gestation (days) Mean number of corpora lutea Mean number of implantations Mean pre-implantation loss (%) Mean number of pups delivered Mean post-implantation loss (%) |
10 22.0 16.2 13.7 16.1 12.3 10.7 |
10 21.9 15.0 14.3 5.6 12.4 13.5 |
8 22.0 16.6 15.9 4.2 13.5 14.4 |
Summary of pup body weight
Sex Dose-level (mg/kg/day) |
0 |
Male 450 |
1000 |
0 |
Female 450 |
1000 |
Body weight (g) PND 1 PND 5 |
8.0 13.5 |
7.8 12.9 |
7.8 12.5 |
7.5 12.7 |
7.5 12.8 |
7.4 12.0 |
Body weight gain (g) PND 1-5 |
+5.4 |
+5.2 |
+4.6 |
+5.1 |
+5.3 |
+4.5 |
PND = post-natal day
> Tissue distribution of cerium:
Tissue distribution analysis of cerium in parental and pup tissues revealed that CeO2 NPs were not detected in almost all of the samples. Only a few samples were slightly above the mean cerium content of blank samples, but it was also observed in vehicle control and there was no correlation in cerium content among the tissues and dose groups.
> Tables
Table 1: Food consumption of CeO2 NPs-treated males and females during the study period.
CeO2 NPs (mg/kg bw/day) |
0 |
100 |
300 |
1000 |
Males |
||||
Pre-mating day 1–4 (g) |
30.6 ± 1.9 |
29.3 ± 0.8 |
28.6 ± 1.5* |
30.5 ± 1.6 |
Pre-mating day 4–8 (g) |
31.3 ± 2.2 |
30.4 ± 1.2 |
30.2 ± 1.6 |
31.2 ± 1.4 |
Pre-mating day 8–11 (g) |
31.4 ± 2.3 |
31.5 ± 1.5 |
30.5 ± 2.3 |
32.2 ± 1.2 |
Pre-mating day 11–14 (g) |
32.3 ± 2.0 |
32.6 ± 1.8 |
31.4 ± 2.4 |
32.7 ± 1.1 |
Total period (g, pre-mating day 1–14) |
31.4 ± 2.0 |
30.9 ± 1.1 |
30.2 ± 1.9 |
31.7 ± 1.0 |
Females |
||||
Pre-mating day 1–4 (g) |
21.1 ± 1.6 |
20.2 ± 1.1 |
21.3 ± 1.1 |
21.2 ± 1.6 |
Pre-mating day 4–8 (g) |
21.8 ± 1.2 |
21.4 ± 0.8 |
22.7 ± 1.2 |
22.2 ± 1.6 |
Pre-mating day 8–11 (g) |
21.9 ± 1.7 |
22.3 ± 1.4 |
23.0 ± 2.2 |
22.1 ± 1.9 |
Pre-mating day 11–14 (g) |
23.0 ± 1.5 |
23.0 ± 1.5 |
23.7 ± 0.8 |
23.3 ± 1.9 |
Gestation day 0–7 (g) |
25.6 ± 1.7 |
26.0 ± 2.6 |
25.7 ± 2.2 |
26.8 ± 1.4 |
Gestation day 7–14 (g) |
25.5 ± 1.5 |
25.9 ± 2.3 |
25.9 ± 2.3 |
25.8 ± 2.0 |
Gestation day 14–20 (g) |
29.8 ± 2.1 |
29.2 ± 1.9 |
29.3 ± 1.9 |
31.5 ± 2.0 |
Post-natal day 0–4 (g) |
35.8 ± 5.0 |
36.8 ± 3.2 |
35.9 ± 6.1 |
40.1 ± 4.1 |
Total period (g, pre-mating day 1 to lactation day 4) |
25.0 ± 1.1 |
25.0 ± 1.4 |
25.4 ± 1.2 |
25.9 ± 1.3 |
*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=12, mean ± SD).
Table 2: Hematology results of CeO2 NPs-treated males and females during the study period.
|
Males |
Females |
||||||
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
0 |
100 |
300 |
1000 |
RBC (106/µL) |
9.1 ± 0.4 |
9.1 ± 0.3 |
8.9 ± 0.5 |
8.9 ± 0.1 |
7.8 ± 0.5 |
7.7 ± 0.3 |
7.9 ± 0.5 |
8.1 ± 0.2 |
HGB (g/dL) |
16.6 ± 0.6 |
16.9 ± 0.4 |
16.8 ± 0.6 |
16.8 ± 0.6 |
14.9 ± 0.9 |
14.8 ± 0.3 |
14.8 ± 0.9 |
15.3 ± 0.5 |
HCT (%) |
51.0 ± 2.5 |
51.4 ± 2.0 |
50.7 ± 2.1 |
50.4 ± 2.1 |
46.0 ± 2.6 |
45.6 ± 1.4 |
45.5 ± 2.7 |
47.0 ± 1.5 |
MCV (fL |
56.1 ± 1.7 |
56.2 ± 0.9 |
57.2 ± 1.8 |
56.9 ± 1.9 |
59.4 ± 0.9 |
59.4 ± 1.2 |
57.9 ± 2.0 |
58.2 ± 1.2 |
MCH (pg) |
18.3 ± 0.4 |
18.5 ± 0.3 |
18.9 ± 0.7 |
18.9 ± 0.5 |
19.3 ± 0.2 |
19.3 ± 0.5 |
18.8 ± 0.7 |
18.9 ± 0.3 |
MCHC (g/dL) |
32.5 ± 0.4 |
33.0 ± 0.7 |
33.1 ± 0.6 |
33.3 ± 0.4 |
32.5 ± 0.5 |
32.5 ± 0.5 |
32.5 ± 0.4 |
32.4 ± 0.4 |
PLT (103/µL) |
1130.8 ± 149.7 |
1024.2 ± 133.7 |
940.4 ± 41.3 |
1101.6 ± 84.8 |
1351.0 ± 167.3 |
1213.2 ± 174.2 |
1258.4 ± 222.7 |
1305.0 ± 253.0 |
RET (%) |
2.5 ± 0.5 |
2.5 ± 0.3 |
2.5 ± 0.3 |
2.5 ± 0.6 |
5.8 ± 1.3 |
6.4 ± 1.7 |
6.3 ± 1.4 |
6.2 ± 1.7 |
RETA (109/µL |
223.8 ± 41.4 |
226.0 ± 22.2 |
223.8 ± 31.6 |
218.4 ± 48.7 |
448.3 ± 78.3 |
492.2 ± 142.9 |
489.7 ± 77.8 |
502.9 ± 136.4 |
WBC(103/µL) |
10.6 ± 3.3 |
11.6 ± 4.1 |
12.4 ± 1.0 |
11.0 ± 2.2 |
9.5 ± 2.5 |
9.8 ± 2.4 |
11.2 ± 1.1 |
12.8 ± 3.2 |
NEU (%) |
14.8 ± 6.9 |
11.4 ± 1.7 |
12.1 ± 3.0 |
13.4 ± 2.5 |
12.2 ± 5.0 |
13.3 ± 2.2 |
12.8 ± 3.2 |
13.2 ± 2.8 |
NEUA(103/µL) |
1.5 ± 0.7 |
1.4 ± 0.7 |
1.5 ± 0.5 |
1.5 ± 0.5 |
1.2 ± 0.7 |
1.3 ± 0.3 |
1.4 ± 0.2 |
1.7 ± 0.8 |
LYM (%) |
81.3 ± 6.7 |
84.7 ± 2.1 |
83.2 ± 3.6 |
81.7 ± 2.0 |
81.5 ± 5.2 |
80.5 ± 2.0 |
80.7 ± 4.0 |
80.3 ± 3.2 |
LYMA(103/µL) |
8.7 ± 2.8 |
9.8 ± 3.3 |
10.3 ± 0.7 |
9.0 ± 1.7 |
7.7 ± 2.0 |
7.9 ± 2.1 |
9.1 ± 1.4 |
10.2 ± 2.3 |
EOS (%) |
0.8 ± 0.2
|
0.9 ± 0.2 |
0.9 ± 0.3 |
1.0 ± 0.4 |
0.8 ± 0.4 |
0.6 ± 0.2 |
0.7 ± 0.3 |
0.7 ± 0.3 |
EOSA (103/µL) |
0.09 ± 0.05 |
0.10 ± 0.03 |
0.12 ± 0.05 |
0.11 ± 0.04 |
0.07 ± 0.02 |
0.06 ± 0.04 |
0.07 ± 0.02 |
0.09 ± 0.04 |
MON (%) |
1.9 ± 0.6 |
1.9 ± 0.6 |
2.3 ± 0.7 |
2.5 ± 0.6 |
4.3 ± 0.7 |
4.0 ± 1.2 |
4.6 ± 0.9 |
4.6 ± 0.8 |
MONA (103/µL) |
0.2 ± 0.1 |
0.3 ± 0.2 |
0.3 ± 0.1 |
0.3 ± 0.0 |
0.4 ± 0.1 |
0.4 ± 0.1 |
0.5 ± 0.1 |
0.6 ± 0.2 |
BAS (%) |
0.6 ± 0.1 |
0.5 ± 0.1 |
0.7 ± 0.1 |
0.6 ± 0.2 |
0.5 ± 0.1 |
0.5 ± 0.2 |
0.4 ± 0.1 |
0.4 ± 0.1 |
BASA (103/µL) |
0.06 ± 0.02 |
0.06 ± 0.03 |
0.09 ± 0.01 |
0.06 ± 0.01 |
0.04 ± 0.01 |
0.05 ± 0.03 |
0.05 ± 0.01 |
0.05 ± 0.02 |
LUC (%) |
0.7 ± 0.3 |
0.6 ± 0.1 |
0.7 ± 0.1 |
0.8 ± 0.1 |
0.8 ± 0.3 |
1.1 ± 0.1 |
0.8 ± 0.3 |
0.8 ± 0.2 |
LUCA (103/µL) |
0.07 ± 0.05 |
0.07 ± 0.04 |
0.09 ± 0.02 |
0.09 ± 0.01 |
0.07 ± 0.01 |
0.10 ± 0.03 |
0.09 ± 0.03 |
0.10 ± 0.03 |
PT (sec) |
14.0 ± 1.7 |
13.7 ± 0.8 |
16.0 ± 2.7 |
13.6 ± 0.7 |
13.6 ± 1.2 |
15.5 ± 0.3* |
15.3 ± 0.9 |
14.4 ± 1.5 |
APTT (sec) |
17.5 ± 0.7 |
18.0 ± 0.8 |
18.3 ± 0.9 |
17.8 ± 1.0
|
6.1 ± 0.7 |
15.6 ± 1.2 |
14.8 ± 1.0 |
15.0 ± 0.9 |
*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=5, mean ± SD).
Table 3: Clinical chemistry results of CeO2 NPs-treated males and females during the study period.
|
Males |
Females |
||||||
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
0 |
100 |
300 |
1000 |
GLU (mg/dL) |
158.9 ± 20.3 |
149.2 ± 28.8 |
157.6 ± 45.1 |
148.5 ± 29.1 |
131.2 ± 22.4 |
116.0 ± 15.1 |
108.8 ± 10.7 |
139.1 ± 17.1 |
BUN (mg/dL) |
15.1 ± 1.6 |
13.7 ± 1.5 |
14.3 ± 1.7 |
13.6 ± 1.8 |
24.1 ± 4.7 |
19.4 ± 3.4 |
21.2 ± 4.1 |
20.3 ± 3.6 |
CREA (mg/dL) |
0.49 ± 0.04 |
0.48 ± 0.03 |
0.47 ± 0.04 |
0.47 ± 0.06 |
0.57 ± 0.08 |
0.51 ± 0.03 |
0.54 ± 0.04 |
0.56 ± 0.03 |
TP (g/dL) |
6.6 ± 0.3 |
6.7 ± 0.2 |
6.6 ± 0.2 |
6.6 ± 0.4 |
7.0 ±0.4 |
7.0 ± 0.4
|
7.0 ± 0.2 |
7.1 ± 0.3 |
ALB (g/dL) |
4.3 ± 0.2 |
4.2 ± 0.1 |
4.2 ± 0.1 |
4.2 ± 0.2 |
4.5 ±0.2 |
4.6 ± 0.3 |
4.5 ± 0.1 |
4.6 ± 0.1 |
A/G (ratio) |
1.8 ± 0.2 |
1.7 ± 0.1 |
1.8 ± 0.1 |
1.8 ± 0.1 |
1.8 ±0.1 |
2.0 ± 0.2 |
1.9 ± 0.1 |
1.9 ± 0.1 |
AST (IU/L) |
149.4 ± 21.9 |
137.5 ± 34.5 |
129.9 ± 4.3 |
153.9 ± 18.5 |
142.7 ± 44.3 |
129.1 ± 23.1 |
135.6 ± 17.7 |
121.3 ± 8.0 |
ALT (IU/L) |
33.4 ± 5.7 |
28.6 ± 5.6 |
32.5 ± 3.0 |
31.5 ± 3.9 |
40.2 ± 8.0 |
41.5 ± 8.1 |
45.1 ± 5.2 |
39.8 ± 2.6 |
TBIL (mg/dL) |
0.14 ± 0.02 |
0.12 ± 0.01 |
0.15 ± 0.02 |
0.13 ± 0.02 |
0.13 ± 0.02 |
0.12 ± 0.02 |
0.13 ± 0.02 |
0.12 ± 0.02 |
GGT (IU/L) |
0.41 ± 0.23 |
0.65 ± 0.13 |
0.60 ± 0.12 |
0.92 ± 0.18** |
0.59 ± 0.22 |
0.78 ± 0.10 |
0.67 ± 0.27 |
0.71 ± 0.38 |
ALP (IU/L) |
581.6 ± 179.2 |
510.3 ± 110.0 |
467.4 ± 57.3 |
514.4 ± 66.4 |
332.3 ± 111.0 |
293.1 ± 58.0 |
232.7 ± 31.7 |
267.1 ± 83.9 |
TCHO (mg/dL) |
70.8 ± 26.9 |
64.2 ± 19.5 |
60.0 ± 8.9 |
68.4 ± 10.2 |
49.8 ± 16.0 |
58.0 ± 16.5 |
53.8 ± 10.8 |
55.2 ± 12.8 |
TG (mg/dL) |
31.8 ± 9.3 |
34.2 ± 11.8 |
23.9 ± 10.0 |
21.4 ± 10.9 |
38.9 ± 22.5 |
36.9 ± 13.1 |
41.7 ± 14.9 |
54.7 ± 25.5 |
Ca (mg/dL) |
11.1 ± 0.3 |
11.3 ± 0.4 |
11.2 ± 0.2 |
11.0 ± 0.7 |
11.7 ± 0.4 |
11.8 ± 0.5 |
11.7 ± 0.3 |
11.9 ± 0.2 |
IP (mg/dL) |
10.2 ± 0.5 |
10.3 ± 0.6 |
10.5 ± 0.4 |
10.3 ± 0.7 |
9.9 ± 0.8 |
10.2 ± 0.1 |
10.3 ± 1.2 |
9.8 ± 0.7 |
K (mmol/L) |
8.3 ± 0.9 |
7.5 ± 0.9 |
8.4 ± 0.9 |
7.8 ± 1.6 |
8.6 ± 0.5 |
8.3 ± 0.5 |
8.2 ± 1.1 |
8.2 ± 1.7 |
CK (IU/L) |
715.6 ± 197.6 |
584.2 ± 203.2 |
483.6 ± 79.4 |
652.0 ± 218.0 |
611.8 ± 331.8 |
518.0 ± 112.9 |
570.6 ± 129.1 |
448.8 ± 130.3 |
PL (mg/dL) |
102.4 ± 25.0 |
93.6 ± 22.7 |
92.0 ± 7.3 |
99.2 ± 10.4 |
106.8 ± 26.9 |
116.8 ± 24.8 |
108.6 ± 14.8 |
115.8 ± 19.1 |
Na (mmol/L) |
148.0 ± 0.7 |
147.8 ± 1.9 |
147.4 ± 1.1 |
147.8 ± 1.6 |
144.8 ± 1.3 |
145.0 ± 1.9 |
145.0 ± 1.4 |
146.0 ± 1.6 |
Cl (mmol/L) |
102.4 ± 1.7 |
101.6 ± 0.6 |
102.6 ± 1.3 |
102.4 ± 0.9 |
101.4 ± 0.6 |
101.2 ± 1.5 |
101.6 ± 1.1 |
102.2 ± 2.4 |
**: Represent a significant difference at the p<0.01 level compared to the vehicle control (n=5, mean ± SD).
Table 4: Absolute and relative organ weights of CeO2 NPs-treated males during the study period.
CeO2 NPs (mg/kg) |
|
0 |
100 |
300 |
1000 |
Terminal body weighta(g) |
(g) |
427.0 ± 38.7 |
439.2 ± 23.7 |
436.4 ± 36.0 |
448.9 ± 28.6 |
N |
12 |
12 |
12 |
12 |
|
Adrenal glands |
Absolute (g) |
0.06 ± 0.01 |
0.07 ± 0.01 |
0.06 ± 0.01 |
0.07 ± 0.00 |
Relativeb(%) |
0.016 ± 0.003 |
0.016 ± 0.002 |
0.016 ± 0.002 |
0.015 ± 0.001 |
|
N |
5 |
5 |
5 |
5 |
|
Brain |
Absolute (g) |
1.97 ± 0.12 |
1.99 ± 0.07 |
2.03 ± 0.09 |
2.00 ± 0.12 |
Relative (%) |
0.484 ± 0.068 |
0.460 ± 0.023 |
0.497 ± 0.030 |
0.461 ± 0.013 |
|
N |
5 |
5 |
5 |
5 |
|
Heart |
Absolute (g) |
1.25 ± 0.12 |
1.29 ± 0.14 |
1.33 ± 0.13 |
1.32 ± 0.11 |
Relative (%) |
0.304 ± 0.023 |
0.300 ± 0.030 |
0.325 ± 0.024 |
0.303 ± 0.028 |
|
N |
5 |
5 |
5 |
5 |
|
Kidneys |
Absolute (g) |
3.25 ± 0.19 |
3.40 ± 0.15 |
3.50 ± 0.33 |
3.47 ± 0.42 |
Relative (%) |
0.794 ± 0.071 |
0.788 ± 0.050 |
0.854 ± 0.047 |
0.797 ± 0.070 |
|
N |
5 |
5 |
5 |
5 |
|
Liver |
Absolute (g) |
11.71 ± 2.07 |
12.74 ± 0.71 |
12.12 ± 2.09 |
12.51 ± 1.41 |
Relative (%) |
2.825 ± 0.217 |
2.950 ± 0.1063 |
2.940 ± 0.246 |
2.870 ± 0.197 |
|
N |
5 |
5 |
5 |
5 |
|
Pituitary gland |
Absolute (g) |
0.01 ± 0.00 |
0.01 ± 0.00 |
0.01 ± 0.00 |
0.01 ± 0.00 |
Relative (%) |
0.003 ± 0.000 |
0.003 ± 0.000 |
0.003 ± 0.000 |
0.003 ± 0.000 |
|
N |
5 |
5 |
5 |
5 |
|
Prostate |
Absolute (g) |
0.62 ± 0.12 |
0.59 ± 0.17 |
0.66 ± 0.13 |
0.65 ± 0.09 |
Relative (%) |
0.144 ± 0.026 |
0.135 ± 0.041 |
0.151 ± 0.031 |
0.146 ± 0.018 |
|
N |
12 |
12 |
12 |
12 |
|
Spleen |
Absolute (g) |
0.66 ± 0.13 |
0.67 ± 0.15 |
0.65 ± 0.13 |
0.69 ± 0.08 |
Relative (%) |
0.158 ± 0.013 |
0.155 ± 0.033 |
0.158 ± 0.020 |
0.158 ± 0.013 |
|
N |
5 |
5 |
5 |
5 |
|
Thymus |
Absolute (g) |
0.34 ± 0.02 |
0.39 ± 0.08 |
0.35 ± 0.11 |
0.32 ± 0.06 |
Relative (%) |
0.083 ± 0.010 |
0.090 ± 0.018 |
0.084 ± 0.019 |
0.072 ± 0.011 |
|
N |
5 |
5 |
5 |
5 |
|
Lungs |
Absolute (g) |
1.50 ± 0.13 |
1.58 ± 0.12 |
1.57 ± 0.17 |
1.62 ± 0.07 |
Relative (%) |
0.367 ± 0.041 |
0.367 ± 0.031 |
0.383 ± 0.021 |
0.374 ± 0.021 |
|
N |
5 |
5 |
5 |
5 |
|
Right testis |
Absolute (g) |
1.70 ± 0.17 |
1.74 ± 0.13 |
1.69 ± 0.11 |
1.65 ± 0.12 |
Relative (%) |
0.400 ± 0.049 |
0.396 ± 0.031 |
0.391 ± 0.051 |
0.369 ± 0.028 |
|
N |
12 |
12 |
12 |
12 |
|
Left testis |
Absolute (g) |
1.73 ± 0.16 |
1.74 ± 0.12 |
1.71 ± 0.11 |
1.67 ± 0.12 |
Relative (%) |
0.407 ± 0.051 |
0.397 ± 0.026 |
0.394 ± 0.044 |
0.372 ± 0.030 |
|
N |
12 |
12 |
12 |
12 |
|
Right epididymis |
Absolute (g) |
0.66 ± 0.06 |
0.69 ± 0.06 |
0.68 ± 0.06 |
0.66 ± 0.07 |
Relative (%) |
0.155 ± 0.017 |
0.158 ± 0.016 |
0.156 ± 0.021 |
0.148 ± 0.021 |
|
N |
12 |
12 |
12 |
12 |
|
Left epididymis |
Absolute (g) |
0.67 ± 0.07 |
0.68 ± 0.06 |
0.66 ± 0.06 |
0.65 ± 0.06 |
Relative (%) |
0.157 ± 0.019 |
0.156 ± 0.014 |
0.152 ± 0.019 |
0.145 ± 0.018 |
|
N |
12 |
12 |
12 |
12 |
|
Seminal vesicles with coagulating glands |
Absolute (g) |
1.68 ± 0.21 |
1.65 ± 0.29 |
1.68 ± 0.22 |
1.69 ± 0.21 |
Relative (%) |
0.396 ± 0.047 |
0.378 ± 0.072 |
0.388 ± 0.057 |
0.377 ± 0.048 |
|
N |
12 |
12 |
12 |
12 |
N=5 or 12, mean ± SD.
a: Terminal body weight were measured immediately before necropsy.
b: Organ weight/terminal body weight ratio.
Table 5: Absolute and relative organ weights of CeO2 NPs-treated females during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
|
Terminal body weight |
(g) |
314.5 ± 19.9 |
316.7 ± 24.1 |
311.0 ± 23.7 |
316.8 ± 19.8 |
Adrenal glands |
Absolute (g) |
0.08 ± 0.01 |
0.07 ± 0.01 |
0.08 ± 0.01 |
0.08 ± 0.01 |
Relative (%) |
0.028 ± 0.003 |
0.024 ± 0.003 |
0.027 ± 0.003 |
0.027 ± 0.002 |
|
Brain |
Absolute (g) |
1.94 ± 0.09 |
1.93 ± 0.08 |
2.02 ± 0.08 |
1.96 ± 0.05 |
Relative (%) |
0.652 ± 0.036 |
0.650 ± 0.022 |
0.692 ± 0.028 |
0.654 ± 0.030 |
|
Heart |
Absolute (g) |
1.00 ± 0.06 |
0.95 ± 0.04 |
0.97 ± 0.07 |
1.04 ± 0.06 |
Relative (%) |
0.334 ± 0.013 |
0.321 ± 0.017 |
0.334 ± 0.026 |
0.346 ± 0.019 |
|
Kidneys |
Absolute (g) |
2.28 ± 0.19 |
2.23 ± 0.21 |
2.14 ± 0.14 |
2.23 ± 0.07 |
Relative (%) |
0.763 ± 0.028 |
0.750 ± 0.051 |
0.734 ± 0.036 |
0.744 ± 0.027 |
|
Liver |
Absolute (g) |
10.52 ± 1.21 |
10.40 ± 0.87 |
10.19 ± 0.30 |
10.91 ± 0.73 |
Relative (%) |
3.522 ± 0.266 |
3.503 ± 0.277 |
3.495 ± 0.169 |
3.638 ± 0.259 |
|
Pituitary gland |
Absolute (g) |
0.02 ± 0.00 |
0.02 ± 0.00 |
0.02 ± 0.00 |
0.02 ± 0.00 |
Relative (%) |
0.006 ± 0.001 |
0.006 ± 0.001 |
0.006 ± 0.001 |
0.006 ± 0.000 |
|
Spleen |
Absolute (g) |
0.62 ± 0.07 |
0.69 ± 0.09 |
0.65 ± 0.08 |
0.70 ± 0.12 |
Relative (%) |
0.209 ± 0.021 |
0.231 ± 0.029 |
0.223 ± 0.032 |
0.232 ± 0.043 |
|
Thymus |
Absolute (g) |
0.31 ± 0.03 |
0.31 ± 0.05 |
0.31 ± 0.08 |
0.38 ± 0.12 |
Relative (%) |
0.104 ± 0.013 |
0.104 ± 0.021 |
0.105 ± 0.027 |
0.125 ± 0.041 |
|
Lungs |
Absolute (g) |
1.34 ± 0.04 |
1.29 ± 0.06 |
1.42 ± 0.05 |
1.36 ± 0.11 |
Relative (%) |
0.451 ± 0.013 |
0.435 ± 0.018 |
0.486 ± 0.022 |
0.453 ± 0.034 |
|
Uterus |
Absolute (g) |
0.78 ± 0.12 |
0.78 ± 0.09 |
0.83 ± 0.12 |
0.73 ± 0.06 |
Relative (%) |
0.260 ± 0.039 |
0.262 ± 0.030 |
0.284 ± 0.035 |
0.243 ± 0.026 |
|
Right ovary |
Absolute (g) |
0.07 ± 0.01 |
0.06 ± 0.01 |
0.06 ± 0.01 |
0.06 ± 0.01 |
Relative (%) |
0.021 ± 0.002 |
0.021 ± 0.004 |
0.019 ± 0.003 |
0.019 ± 0.004 |
|
Left Ovary |
Absolute (g) |
0.07 ± 0.02 |
0.06 ± 0.01 |
0.06 ± 0.01 |
0.05 ± 0.01 |
Relative (%) |
0.023 ± 0.006 |
0.020 ± 0.002 |
0.020 ± 0.005 |
0.018 ± 0.002 |
|
N=5, mean ± SD.
Table 6: Fertility with precoital time results of CeO2 NPs treated males during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
Males |
||||
Mating indexa |
100 |
100 |
100 |
100 |
Fertility indexb |
100 |
100 |
100 |
100 |
Fecundity indexc |
100 |
100 |
100 |
100 |
Females |
||||
Mating indexd |
100 |
100 |
100 |
100 |
Fertility indexe |
100 |
100 |
100 |
100 |
Pregnancy indexf |
100 |
100 |
100 |
100 |
Precoital Time (day) |
3.3 ± 3.6 |
1.8 ± 0.6 |
2.2 ± 1.1 |
1.9 ± 1.1 |
N=12, mean ± SD.
a: (No. of males with evidence of mating/No. of males paired) x 100.
b: (No. of males impregnating a female/No. of males paired) x 100.
c: (No. of males impregnating a female/No. of males with evidence of mating) x 100.
d: (No. of females with evidence of mating/No. of females paired) x 100.
e: (No. of pregnant females/No. of females paired) x 100.
f: (No. of pregnant females/No. of females with evidence of mating) x100.
Table 7: Reproductive and litter findings results of CeO2 NPs-treated females during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
Gestation period (day) |
21.5 ± 0.4 |
21.5 ± 0.3 |
21.6 ± 0.3 |
21.8 ± 0.4 |
Corpora lutea (N) |
17.8 ± 2.8 |
16.5 ± 2.6 |
16.3 ± 1.7 |
17.4 ± 2.0 |
Implantations (N) |
15.3 ± 3.1 |
15.3 ± 2.4 |
14.8 ± 2.1 |
15.4 ± 2.0 |
Pups born (N) |
14.8 ± 3.1 |
14.4 ± 2.4 |
13.9 ± 2.0 |
14.8 ± 2.1 |
Perinatal death (N) |
0.08 ± 0.29 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.25 ± 0.45 |
Unaccounted-for sitesa(%) |
2.8 ± 4.5 |
5.5 ± 5.8 |
6.2 ± 4.5 |
3.9 ± 4.5 |
Sex ratiob(%) |
106.9 ± 62.6 |
93.9 ± 28.2 |
105.3 ± 55.5 |
130.7 ± 66.8 |
Live litter size (N) PND 0 PND 4 |
14.8 ± 3.1 14.8 ± 3.1 |
14.4 ± 2.4 14.3 ± 2.4 |
13.9 ± 2.0 13.8 ± 2.0 |
14.6 ± 2.1 14.4 ± 2.0 |
Viability indexc(%) |
100.0 |
99.4 ± 1.9 |
98.9 ± 2.6 |
99.0 ± 2.4 |
Delivery indexd(%) |
100.0 |
100.0 |
100.0 |
100.0 |
Pups with external abnormalities |
0 |
0 |
0 |
0 |
N = 12, mean ± SD.
a: (No. of implantation sites/litter) - (No. of live pups at birth/litter)/No. of implantation sites/litter x 100.
b: (No. of male pups on PND 0/litter)/(No. of female pups on PND 0/litter) x 100.
c: (No. of live pups on PND 4/litter)/(No. of live pups at birth/litter) x 100.
d: (No. of dams with live pups)/(No. of pregnant dams) x 100.
Table 8: F1 pups body weights of CeO2 NPs-treated parental animals during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
F1 male pups |
||||
PND 0 |
||||
Body weight (g) |
6.5 ± 0.4 |
6.7 ± 0.3 |
6.8 ± 0.6 |
7.0 ± 0.5 |
Covariate-adjusted mean (g) |
6.5 |
6.7 |
6.8 |
7.0* |
PND 4 |
||||
Body weight (g) |
10.1 ± 0.8 |
10.7 ± 1.1 |
11.0 ± 1.1 |
11.0 ± 1.2 |
Covariate-adjusted mean (g) |
10.1 |
10.6 |
10.9 |
11.2* |
F1 female pups |
||||
PND 0 |
||||
Body weight (g) |
6.2 ± 0.4 |
6.3 ± 0.3 |
6.4 ± 0.6 |
6.7 ± 0.6 |
Covariate-adjusted mean (g) |
6.2 |
6.3 |
6.4 |
6.7* |
PND 4 |
||||
Body weight (g) |
9.5 ± 0.9 |
10.0 ± 1.0 |
10.4 ± 1.2 |
10.6 ± 1.4 |
Covariate-adjusted mean (g) |
9.6 |
10.1 |
10.4 |
10.5 |
N =12, mean ± SD.
*Represent a significant difference at the p<0.05 level compared to the vehicle control.
Table 3a: Body weight and body weight change (F0 generation)
Sex |
Male |
Female |
||||||
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
0 |
150 |
450 |
1000 |
Premating |
|
|
|
|
|
|
|
|
Days 1 - 71 |
+317 |
+331 |
+312 |
+331 |
+139 |
+144 |
+156** |
+147 |
Days 1 - 127 |
+396 |
+416 |
+395 |
+416 |
/ |
/ |
/ |
/ |
Gestationa |
|
|
|
|
|
|
|
|
GD 0 - 20 |
/ |
/ |
/ |
/ |
+144 |
+151 |
+143 |
+150 |
Lactation |
|
|
|
|
|
|
|
|
LD 1 - 21 |
/ |
/ |
/ |
/ |
+16 |
+6 |
+18 |
+18 |
GD: gestation day, LD: lactation day, /: not applicable, a: only includes pregnant females with live fetuses.
Statistically significant **: p<0.01.
Table 3b: Body weight and body weight change (F1 generation)
Sex |
Male |
Female |
||||||
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
0 |
150 |
450 |
1000 |
Premating |
|
|
|
|
|
|
|
|
. Days 1 - 71 |
+462 |
+473 |
+477 |
+475 |
+222 |
+232 |
+226 |
+222 |
. Days 1 - 127 |
+566 |
+585 |
+594 |
+581 |
/ |
/ |
/ |
/ |
Gestation |
|
|
|
|
|
|
|
|
. GD 0 - 20 |
/ |
/ |
/ |
/ |
+148 |
+161 |
+156 |
+166 |
Lactation |
|
|
|
|
|
|
|
|
. LD 1 - 21 |
/ |
/ |
/ |
/ |
+19 |
+13 |
+17 |
+23 |
GD: gestation day, LD: lactation day, /: not applicable.
Table 4a : Estrous cycles (F0 generation)
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
Mean number of cycles per female |
4.7 |
4.8 |
4.8 |
4.7 |
Mean cycle length (days) |
4.0 |
4.0 |
4.1 |
4.1 |
Number of abnormally cycling females |
1 |
2 |
2 |
2 |
An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.
Table 4b : Estrous cycles (F1 generation)
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
Mean number of cycles per female |
4.6 |
4.5 |
4.8 |
4.6 |
Mean cycle length (days) |
4.2 |
4.4 |
4.1 |
4.2 |
Number of abnormally cycling females |
1 |
2 |
1 |
2 |
An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.
Table 5a: Mating, fertility and parturition (F0 generation)
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
Number of males + females paired |
25 +25 |
25 + 25 |
25 + 25 |
25 + 25 |
Number of pairs mated |
25 |
25 |
25 |
25 |
Mating index |
100% |
100% |
100% |
100% |
Mean number of days taken to mate |
2.0 |
2.9 |
3.4 |
3.0 |
Number of pregnant females |
25 |
24 |
24 |
23 |
Fertility index |
100% |
96% |
96% |
92% |
Number of females delivering live pups |
25 |
24 |
24 |
22 |
Gestation index |
100% |
100% |
100% |
96% |
Mean duration of gestation (days) |
22.0 |
21.8 |
21.8 |
21.9 |
Table 5b: Mating, fertility and parturition (F1 generation)
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
Number of males paired |
25 |
25 |
24 |
24 |
Number of males mated |
25 |
23 |
24 |
24 |
Male fertility index |
100% |
92% |
100% |
100% |
Number of females paired |
25 |
25 |
24 |
24 |
Number of females mated |
25 |
25 |
24 |
24 |
Mating index |
100% |
100% |
100% |
100% |
Mean number of days taken to mate |
3.0 |
4.3 |
2.7 |
2.8 |
Number of pregnant females |
24 |
21 |
22 |
22 |
Fertility index |
96% |
84% |
92% |
92% |
Number of females delivering live pups |
23a |
21 |
22 |
20a |
Gestation index |
96% |
100% |
100% |
91% |
Mean duration of gestation (days) |
21.9 |
21.9 |
21.9 |
21.9 |
a: females sacrificed mid-parturition because of poor clinical condition.
Table 6a: Mating, fertility and parturition (F0 generation) Cont’
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
Mean number of implantation sites |
13.8 |
14.8 |
14.4 |
14.1 |
Mean number of pups born |
11.8 |
12.7 |
12.7 |
12.3 |
Post-implantation loss (calculated manually) |
14.2 |
14.2 |
11.7 |
12.4 |
Number of entire litters dead (no. of pups) |
1 (14) |
0 |
2 (24) |
1 (14) |
Number of dead pups: days 1 - 4 |
22 |
6** |
38* |
34*a |
Number of dead pups: days 5 - 21 |
0 |
1 |
0 |
1 |
Number of litters with dead pups |
6 |
4 |
6 |
11 |
% of male pups at birth |
51.7% |
46.7% |
48.8% |
50.0% |
Statistically significant *: p<0.05, **: p<0.01. a: does not include one pup which was cannibalized and could not be sexed.
Table 6b: Mating, fertility and parturition (F1 generation) Cont’
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
Mean number of implantation sites |
12.1 |
14.4 |
13.1 |
14.4 |
Mean number of pups born |
11.1 |
12.3 |
12.0 |
12.1 |
Post-implantation loss (calculated manually) |
10.1 |
14.0 |
8.2 |
16.1 |
Number of entire litters dead |
1 |
0 |
0 |
0 |
Number of dead pups: days 1 - 4 |
19 |
12 |
12 |
14 |
Number of dead pups: days 5 - 21 |
1 |
4 |
1 |
0 |
Number of litters with dead pups |
11 |
9 |
5 |
8 |
% of male pups at birth |
46.6 |
50.8 |
39.5 |
51.4 |
Table 7:Relevant changes in mean absolute and relative organ weights in treated F0 parents (% changes from controls)
Sex |
Male |
Female |
||||
Group |
2 |
3 |
4 |
2 |
3 |
4 |
Dose-level (mg/kg/day) |
150 |
450 |
1000 |
150 |
450 |
1000 |
Exam. animals / Num. of animals |
25/25 |
25/25 |
25/25 |
25/25 |
23/25 |
24/25 |
Body weight |
+3 |
-1 |
+3 |
0 |
+2 |
+2 |
- Kidney, left |
|
|
|
|
|
|
. absolute |
+6* |
+7* |
+12** |
+2 |
+6* |
+3 |
. relative |
+3 |
+7* |
+9** |
+2 |
+3 |
+1 |
- Kidney, right |
|
|
|
|
|
|
. absolute |
+3 |
+6* |
+10** |
+5 |
+5 |
+4 |
. relative |
0 |
+7** |
+7** |
+4* |
+3 |
+2 |
- Liver |
|
|
|
|
|
|
. absolute |
+10** |
+9** |
+17** |
+2 |
+4 |
+1 |
. relative |
+7** |
+10** |
+14** |
+2 |
+1 |
-1 |
- Ovary, left |
|
|
|
|
|
|
. absolute |
|
|
|
+16* |
+14 |
+17* |
. relative |
|
|
|
+16 |
+11 |
+15* |
- Ovary, right |
|
|
|
|
|
|
. absolute |
|
|
|
+9 |
+12 |
+18** |
. relative |
|
|
|
+9 |
+10 |
+16* |
Statistically significant from controls: *: p<0.05, **: p<0.01.
Statistical significance determined for organ weights values and not percent changes.
Table 8:Relevant changes in mean absolute and relative organ weights in treated F1 parents (% changes from controls)
Sex |
Male |
Female |
||||
Group |
2 |
3 |
4 |
2 |
3 |
4 |
Dose-level (mg/kg/day) |
150 |
450 |
1000 |
150 |
450 |
1000 |
Exam. animals / Num. of animals |
24/25 |
25/25 |
25/25 |
25/25 |
24/25 |
22/25 |
Body weight |
+4 |
+5 |
+3 |
+4 |
+1 |
+2 |
- Kidney, left |
|
|
|
|
|
|
. absolute |
+8* |
+10** |
+9** |
+3 |
-2 |
+4 |
. relative |
+4 |
+5 |
+7* |
-1 |
-3 |
+2 |
- Kidney, right |
|
|
|
|
|
|
. absolute |
+7 |
+10** |
+6* |
+3 |
0 |
+5 |
. relative |
+3 |
+5 |
+4 |
-1 |
-2 |
+2 |
- Liver |
|
|
|
|
|
|
. absolute |
+8* |
+9* |
+13** |
+1 |
+2 |
0 |
. relative |
+4 |
+4 |
+10** |
-3 |
+1 |
-2 |
Statistically significant from controls: *: p<0.05, **: p<0.01.
Statistical significance determined for organ weights values and not percent changes.
Table 9a : Sperm analysis (F0 generation)
Dose-level (mg/kg/day) |
0 |
1000 |
Mean number of epididymal sperm (106/cauda) |
171 |
173 |
% of motile sperm |
96 |
93 |
% of morphologically normal sperm |
96 |
96 |
Mean number of sperm heads (106/g testis) |
125 |
125 |
Table 9b : Sperm analysis (F1 generation)
Dose-level (mg/kg/day) |
0 |
1000 |
Mean number of epididymal sperm (106/cauda) |
177 |
191 |
% of motile sperm |
90 |
99 |
% of morphologically normal sperm |
83 |
92 |
Mean number of sperm heads (106/g testis) |
122 |
121 |
Table 10: Body weight and body weight change in F1 generation during lactation (pups)
Sex |
Male |
Female |
||||||
Dose-level (mg/kg/day) |
0 |
150 |
450 |
1000 |
0 |
150 |
450 |
1000 |
Body weight (g) |
|
|
|
|
|
|
|
|
. Day 1 |
7.8 |
7.7 |
7.5 |
7.6 |
7.3 |
7.3 |
7.1 |
7.1 |
. Day 21 |
57.1 |
60.6 |
58.6 |
57.8 |
56.1 |
58.5 |
56.7 |
56.3 |
Body weight gain (g) |
|
|
|
|
|
|
|
|
. Days 1 - 21 |
+49.3 |
+52.9 |
+51.1 |
+50.2 |
+48.8 |
+51.2 |
+49.6 |
+49.2 |
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
No studies are available for the reaction mass of cerium dioxide and zirconium dioxide. However, due to similar physicochemical, toxicological, ecotoxicological and environmental properties, data on/representative for the reaction mass' constituents cerium dioxide and zirconium dioxide are considered to conclude on the reproductive toxicity of the reaction mass of cerium dioxide and zirconium dioxide.
Reproductive toxicity studies on the reaction mass of cerium dioxide and zirconium dioxide are therefore not regarded as scientifically necessary according to section 1 of Reach Annex XI and are not recommended under animal protection considerations.
Cerium dioxide
Bulk
The potential reproductive toxicity of bulk cerium dioxide was tested in rats in an OECD TG 422 GLP-compliant study (Klimisch 1) following daily oral gavage from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at dose levels up to 1000 mg/kg bw/day (Davies, 2010a). There were no relevant differences from control animals for pairing, mating, fertility and delivery parameters. Macroscopic and microscopic examinations at necropsy did not reveal any treatment-related findings or organ weight changes in reproductive organs. The NOEL for reproductive performance was therefore >= 1000 mg/kg bw/day, a limit dose for repeated-dose toxicity, illustrating the absence of reproductive toxicity of bulk cerium dioxide.
Furthermore, there were no adverse effects on the reproductive organs of rats exposed by inhalation for 90 days to bulk cerium dioxide up to the very high concentration of 507.5 mg/m3 (Viau, 1994; see section '7.5.3 Repeated dose toxicity: inhalation' for details). There were no relevant changes in the weight of gonads, prostate or uterus. No macroscopic abnormalities of the reproductive organs were observed at necropsy. There were no relevant histopathological findings at the microscopic examination of epididymides, testes, prostate, ovaries or uterus in any group.
Nano
First, the effects of nano cerium dioxide on the general toxicity and reproductive or developmental toxicity was evaluated, in a reproduction/developmental toxicity screening study, following daily oral administration by gavage to Sprague-Dawley rats (Lee et al., 2020; Klimisch 1). The study was performed according to OECD guideline 422 and was compliant to GLP. Groups of 12 male and 12 female Sprague-Dawley rats were treated by gavage with the test substance at dose levels of 0 (controls, vehicle), 100, 300 and 1000 mg/kg bw/day nano cerium dioxide in water from 2 weeks before mating, through mating and, for the females, through gestation until lactation day 4, corresponding to 38 days of treatment in males and 41 days of treatment in females. Effects of the treatment on mortality, clinical signs, body weight and body weight gain, food consumption, functional observation battery, hematology and chemical chemistry were evaluated. All animals were sacrificed at the end of the study and gross necropsy and histopathology was performed. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Precoital time and fertility-related data, including mating, fertility, fecundity, pregnancy index and delivery index were calculated. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups were evaluated. Pup mortality, viability index, individual body weight and general clinical signs were examined once daily. Further, parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected for cerium content analysis using inductively coupled plasma mass spectrometry (ICP-MS).
No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. No effect of treatment was observed in hematology and clinical biochemistry analyses and in the functional observational battery results. The treatment with the test substance induced no change in organ weight and no gross or histopathological lesion in this study.
No estrus cycle abnormalities were observed in females and no treatment-related changes in fertility results with precoital time were found. No effect of the treatment was observed on the mating index, fertility index, fecundity index and pregnancy index. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods. Pups showed no effect of treatment on survival, clinical signs, body weight and body weight gain. No pup with external abnormalities was found in this study.
Tissue distribution analysis of cerium in parental and pup tissues revealed that nano cerium dioxide was not detected in almost all of the samples. Only a few samples were slightly above the mean cerium content of blank samples, but it was also observed in vehicle control and there was no correlation in cerium content among the tissues and dose groups.
The authors concluded that, under the experimental conditions of this study, no nano cerium dioxide-related adverse effects on the general systemic signs as well as on the reproductive performance or developmental toxicity was observed at doses up to and including at 1000 mg/kg bw/day. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at >= 1000 mg/kg bw/day and the NOAEL for developmental effects in the pups can be set at >= 1000 mg/kg bw/day. In addition, cerium dioxide nanoparticles were not deposited in the parental or pup internal organs after repeated oral exposure.
Further, in a reproduction/developmental toxicity screening test, performed similarly to OECD guideline 421 and following the principles of GLP (but not audited by the Quality Assurance unit since this study was used as a dose-range finding test for the 2-generation study, see below), the potential general toxicity and reproductive or developmental toxicity of an organo-metallic nanoform of cerium dioxide (cerium and iron oxide isostearate) was tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at dose levels of 0, 450 or 1000 mg/kg bw/day in corn oil (Davies, 2010b).
No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose levels. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.
Pups showed no effects of treatment on survival.
Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg bw/day (-15% in the males and -12% in the females, not statistically significant). This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded.
It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio. There were no treatment-related macroscopic abnormalities.
Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg bw/day; however, the pups of the animals treated at 1000 mg/kg bw/day did have a lower mean body weight gain from post-natal days 1 to 5, but these effects were not seen in the 2-generation reproduction study summarised below.
Then, the potential of the same organo-metallic nanoform of cerium oxide to induce effects on reproduction was assessed in a 2-generation reproduction study performed according to OECD guideline 416 and in accordance with GLP (Spézia, 2011). This study was scored as validity 1 according to Klimisch criteria and thus was considered as the key study for this endpoint.
In this study, four groups of 25 male and 25 female Sprague-Dawley rats constituting the F0 parents received the test item or the vehicle only, daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. The test item was administered as a suspension in corn oil, by oral gavage, at dose levels of 0 (control, vehicle only), 150, 450 or 1000 mg/kg bw/day, under a constant dosage volume of 4 mL/kg/day. After weaning (day 22 post-partum) of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of four groups of 25 male and 25 female rats. Three groups received the test item daily for 10 weeks prior to mating and, during mating, gestation and lactation until weaning of the pups. The test item was administered as a suspension in corn oil, by oral gavage, at dose-levels of 150, 450 or 1000 mg/kg bw/day, under a constant dosage volume of 4 mL/kg/day. Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group.
The F0 and F1 animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once a week. Body weight and food consumption were recorded weekly.
The estrous cycles of both F0 and F1 females were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days for F0 females and up to 16 days for F1 females. The females of both generations were allowed to litter and rear their progeny until weaning. The F1 and F2 pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening and, surface righting, cliff avoidance and air righting reflexes).
Furthermore, each animal of the F1 generation was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight was recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, and at 9 weeks of age, spontaneous locomotor activity was measured using automated infrared sensor equipment.
After weaning of the pups, the males and females of both the F0 and F1 generation were sacrificed.
Sperm analysis was performed on the first ten F0 and F1 males of the control and high dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females of both generations, and designated organs were weighed. A microscopic examination was performed on the reproductive organs of the control and high dose group animals and on all macroscopic lesions.
In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then, submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.
In the F0 generation, there were no animals found dead. A total of four females (one at 1000 mg/kg bw/day, two at 450 mg/kg bw/day and one in controls) were prematurely sacrificed during lactation, and microscopic examination findings excluded a relationship to the test item.
There were no treatment-related effects on body weight or body weight gain and no test item treatment-related clinical signs were observed. There was a slight statistically significant increase in mean food consumption in males and females treated at 1000 mg/kg bw/day during the pre-mating period and mid-gestation and in females treated at 450 mg/kg bw/day at the beginning of the pre-mating period. There were no effects of treatment with the test item on food consumption during the lactation period.
There were no effects on estrous cyclicity, mating or fertility parameters at any dose level; however, pup mortality was statistically significantly higher at 450 and 1000 mg/kg bw/day. One female at 1000 mg/kg bw/day and one female at 450 mg/kg bw/day had clinical signs that could indicate poor maternal nesting or nursing behaviour. Few of the dead or cannibalised pups from the other litters had clinical signs. Since the majority of the found dead pups were concentrated in a few litters, it is considered unlikely that their deaths were related directly to test item treatment; instead, it is more probably related to poor maternal nesting/nursing behaviour. The presence of one dead litter in the control group suggests that the dead litters were incidental to treatment with the test item.
No effects on spermatogenesis were detected after analysis of epididymidal sperm motility and morphology and count and, after analysis of testicular sperm count. At histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
At all dose levels, there were a few organ weight changes that were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose related in magnitude, and/or were not consistent for the sexes.
There were no test item‑related microscopic findings in the genital organs of F0 parents.
There was a statistically significant increase in the mean ovary weight in F0 females treated at 1000 mg/kg bw/day, correlating with an increase in the mean number of corpora lutea. This finding was not considered to be toxicologically significant based on the direction of the change. In this group, the mean number of primordial follicles was slightly decreased. Since such variation was not found in F1 females, this variation was not considered to be of toxicological significance.
The F1 generation showed no effects of treatment while pups; there were no test item treatment‑related clinical signs, no effects on body weight and no differences in physical or reflex development when compared with the controls and no treatment-related findings were observed at external examination of F1 pups.
There were no test item treatment-related mortalities.
As for the F0 generation, the males treated at 450 or 1000 mg/kg bw/day and the females treated at 1000 mg/kg bw/day had statistically significantly increased food consumption which correlated with a tendency towards a non-statistically significant increase in body weight gains. The females treated at 150 or 450 mg/kg bw/day also had slightly increased food consumption during lactation.
There were no effects on estrous cyclicity or mating.
Pup mortality was not higher in test item-treated groups than in the controls.
No effects on spermatogenesis were detected after analysis of epididymidal sperm motility, morphology or count or testicular sperm count. At histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
The test item administration was associated with increases in mean kidney and liver weights in F1 males. These changes were not observed in the females. However, these changes were not considered to be related to the test item, as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
There were no test item-related macroscopic findings in F1 parents. There were no test item‑related microscopic findings in the genital organs from F1 parents.
There were increases in the mean number of corpora lutea in high-dose F1 females. This finding was not considered to be toxicologically significant based on the direction of the change. There were no significant differences in the mean number of primordial follicles between control and high‑dose F1 females.
In the F2 generation pups, there were no test item treatment-related clinical signs and no effects on physical or reflex development.
The pups from the groups treated at 150 mg/kg bw/day had statistically significantly higher mean body weight gains at mid-lactation but there were no effects at 450 or 1000 mg/kg bw/day. Therefore, a relationship to the treatment with the test item was considered unlikely.
There were no test item-related changes in organ weights or macroscopic findings in the pups.
From the results observed in this study, it can be concluded that the No Observed Adverse Effect Level of the test substance for reproductive performance and systemic toxicity of F0 and F1 generations was considered to be >= 1000 mg/kg bw/day.
Furthermore, the test substance did not cause delayed or impaired development in the F1 or F2 generations following treatment of the parents and thus, the No Observed Effect Level (NOEL) for peri- and post-natal development of F1 and F2 generations was considered to be >= 1000 mg/kg bw/day.
Finally, no effects on reproductive organs were observed in the short-term repeated dose inhalation toxicity study of Landsiedel et al. (2014) up to10 mg/m3 nano cerium dioxide after 5 days of exposure, which can be considered as supporting information.
Zirconium dioxide
A Klimisch 1 study performed with the read across substance (bulk) zirconium acetate, i.e. a 'water soluble' zirconium compound which can be considered representative for other zirconium compounds including zirconium dioxide, is included for the reproductive/developmental toxicity screening endpoint.
The systemic toxic effects of the read across substance zirconium acetate after repeated oral dosing, as well as any toxic effects on reproduction and development, were investigated in Sprague Dawley rats up to early lactation (day 4 post partum) by Rossiello (2013). The study was performed according to OECD guideline 422 and under GLP principles.
Three groups of 10 males and 10 females each received the test item, by oral gavage, at 100, 300 and 1000 mg anhydrous zirconium acetate/kg bw/day. A similar constituted control group received the vehicle alone during the treatment period. The overall dosing period was 32 days for males, which included 2 weeks before pairing and continuously thereafter up to the day before necropsy, and up to 50 days for females, including 2 weeks before pairing and thereafter during pairing, gestation and lactation periods until day 3 post partum.
The parental animals were followed for daily clinical signs, weekly body weight, food consumption, neurotoxicity assessment, oestrous cycle, mating performance, clinical pathology evaluation including haematology and clinical chemistry, and offspring delivery. A detailed macroscopic examination, determination of organ weights, and histopathological examination, including the spermatogenic cycle, were performed. Pups were also checked for sex, body weight, clinical signs and macroscopic observations.
No mortality occurred in the study. No treatment related findings were observed either during the in vivo phase or at post mortem examination of parent animals. Microscopically, a treatment related finding was seen in males receiving 300 and 1000 mg zirconium acetate/kg bw/day consisting of minimal focal vacuolation of squamous epithelium (limiting ridge) of the non-glandular region of the stomach. This change may be attributed to a local irritant effect of the compound administered by oral gavage and since humans do not have a forestomach or structural analogue to the forestomach, this finding is not considered of toxicological relevance. In addition, no abnormalities were found during the evaluation of the spermatogenic cycle. No treatment related effects were observed in the number of oestrous cycle, pre-coital intervals, copulatory and fertility indices between treated and control groups. No significant differences were observed in the number of implantations, corpora lutea, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated groups.
No effects were noted on reproduction and development at any dose. On the basis of the results obtained in this study, the NOAEL for reproduction/developmental toxicity was considered to be >= 1000 mg/kg bw/day (expressed as anhydrous zirconium acetate), i.e., the highest dose tested.
Further, no effect of zirconium dioxide was observed on male gonads of several species (cats, rats, dogs, guinea-pigs, rabbits) after repeated exposure by inhalation to a concentration level up to 100.8 mg/m3 (Spiegl et al., 1956; see section '7.5.3 Repeated dose toxicity: inhalation' for details).
Reaction mass of cerium dioxide and zirconium dioxide
Based on all information discussed above, it can be safely concluded that the reaction mass of cerium dioxide and zirconium dioxide, regardless its nano-status, is not expected to adversely affect reproduction/development.
Toxicity to reproduction |
Reaction mass |
Bulk cerium dioxide |
Nano cerium dioxide (or organometallic nanoform of cerium dioxide (cerium and iron oxide isostearate) |
Bulk zirconium dioxide |
Bulk zirconium acetate |
Effects on fertility/ development |
- |
Screening for reproductive/developmental toxicity (OECD 422, GLP): NOEL >= 1000 mg/kg bw/d |
Screening for reproductive/developmental toxicity (OECD 422, GLP): NOAEL (parent) >= 1000 mg/kg bw/day NOEL (developmental) >= 1000 mg/kg bw/day 2-generation study by gavage (OECD TG 416, GLP): NOAEL (parental F0/F1) >= 1000 mg/kg bw/day NOEL (offspring F1/F2) >= 1000 mg/kg bw/day |
- |
NOAEL >= 1000 mg/kg bw/d |
Further information |
- |
No effect on reproductive organs up to 507.5 mg/m3 from sub-chronic repeated dose inhalation toxicity study |
No effect on reproductive organs up to10 mg/m3 from 5 day short-term inhalation toxicity study |
No effect on reproductive organs up to 100.8 mg/m3 from repeated dose inhalation toxicity study |
|
Effects on developmental toxicity
Description of key information
In the absence of (reliable) information on the reaction mass of cerium dioxide and zirconium dioxide itself, the endpoint was covered using a weight-of-evidence approach using the results of an OECD 422 study in rats (Lee et al., 2020) as well as the results of a study in mouse performed according to a method similar to OECD guideline 414 (Campagnolo and Pietroiusti, 2015). Both studies were performed using nano cerium dioxide. In the study performed by Lee et al. (2020), the oral repeated exposure of the rats to nano cerium dioxide did not result in adverse effects on development up to and including at the highest dose tested, i.e. 1000 mg/kg bw/day. In the study performed by Campagnolo and Pietroiusti (2015), no systemic maternal toxicity nor embryotoxicity in foetuses was observed in mice exposed via pharyngeal aspiration to a dose of 20 mg/kg bw (i.e. the highest dose tested).
Based on an assessment taking into account all available data including toxicokinetic considerations, it is considered scientifically not necessary nor justifiable to perform further developmental toxicity studies with the reaction mass of cerium dioxide and zirconium dioxide or either of its constituents.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- Read across based on studies performed with nano cerium dioxide. The read across justification document is attached to IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause adverse effects on development. Conclusion based on the results of read across studies performed with nano cerium dioxide.
- Key result
- Abnormalities:
- no effects observed
- Key result
- Dose descriptor:
- other: read across conclusion
- Remarks on result:
- other: The reaction mass of cerium dioxide and zirconium dioxide was concluded not to cause any adverse effects on development. Conclusion based on the results of read across studies performed with nano cerium dioxide.
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Endpoint:
- developmental toxicity
- Data waiving:
- other justification
- Justification for data waiving:
- other:
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
In the absence of available data on the reaction mass with cerium dioxide and zirconium dioxide, this justification uses all relevant available information on the constituents of the reaction mass, i.e. cerium dioxide and zirconium dioxide.
Cerium dioxide
- Nano and bulk forms of cerium dioxide have shown no systemic toxicity and no effect on the reproductive organs and function as well as no pre- and post-natal toxicity and no teratogenic effects in 5 studies performed either by oral or pulmonary (pharyngeal aspiration) routes, up to the highest dose tested in these studies (1000 mg/kg bw/day in oral studies and 20 mg/kg bw/day in the pulmonary study) (bulk CeO2: OECD 422, Davies, 2010a; nano CeO2: OECD 422, Lee et al., 2020 and cfr. OECD 414, Campagnolo and Pietroiusti, 2015; nano cerium and iron oxide isostearate: OECD 421, Davies, 2010b and OECD 416, Spézia, 2011). More particularly, the two OECD 422 studies performed with bulk (Davies, 2010a) and nano CeO2 (Lee et al., 2020) did not evidence difference in effect between the two forms tested. Indeed, no systemic toxicity and no reproductive/developmental effects were observed in both studies. This is likely due to agglomeration/aggregation of these insoluble CeO2 forms (bulk: < 0.123 µg/L at 20°C, pH: 6.01-6.41 (Weissenfeld, 2007; OECD 105); nano: 1.76 µg/L at 20°C, pH 7.8 (enhanced OECD 105, 2019)) which induced a similar tissue distribution, as previously observed by Geraerts et al. (2012) and Gosens et al. (1994). Indeed, these authors have also reported a lack of difference in systemic distribution and effects for bulk and nano CeO2. Further, the absence of evidence of novel toxicological properties of globular biodurable nanomaterials in comparison to their micromaterial counterparts was also evidenced by Moreno-Horn and Gebel (2014) and NANoREG (2017).
- No tissue distribution of nanoceria was found in the OECD 422 study after exposure of the rats by oral route (Lee et al., 2020). These results are supported by other studies performed in rats and mice (e.g. He et al., 2010; Hirst et al., 2011; Molina et al., 2014; Park et al., 2009). Furthermore, rather low to negligible systemic dissemination and no exposure-related lesions were detected in the organs (apart from the respiratory tract and its lymph nodes) investigated after exposure by inhalation of rats to nano CeO2 for 24 months in a carcinogenicity study performed according to OECD guideline 453. In addition, no particles were visible microscopically in any of these organs (Tentschert et al., 2020; Schaudien et al., 2019; Laux, 2017). These results are in accordance with previous results observed by Geraerts et al. (2012), Keller et al. (2014a,b) and Gosens et al. (2014), who reported low systemic distribution and no related morphological abnormalities in the extrapulomonary organs studied after exposure to up to 28 days to 2 nanosized materials of CeO2 and one microsized material of CeO2 in rats.
- Further, the available studies have used 2 rodents species: four of these studies (bulk CeO2: OECD 422, Davies, 2010a; nano CeO2: OECD 422, Lee et al., 2020; nano cerium and iron oxide isostearate: OECD 421, Davies, 2010b and OECD 416, Spézia, 2011) were performed in the rat and the fifth one (nano CeO2: equivalent to OECD 414, Campagnolo and Pietroiusti, 2015) used mice. No difference in systemic toxicity of cerium dioxide was seen in these two rodents species. Moreover, in the teratogenicity study performed in mice that received pharyngeal aspiration of nano CeO2 at up to 20 mg/kg bw/day, the authors found no overt toxicity in terms of miscarriage or malformations (Campagnolo and Pietroiusti, 2015). The authors concluded that their data suggest a lack of serious embryotoxicity after pulmonary exposure to cerium dioxide and they claimed that, from the perspective of regulators and policy makers, their data imply that unintended pulmonary exposure to cerium dioxide nanoparticles at doses which can be realistically expected in occupational and environmental settings should not pose peculiar risk to pregnant women. In addition, no further developmental toxicity is expected in rabbits, as it has been shown by Braakhuis et al. (2019) that rats and rabbits do not differ in sensitivity to developmental effects.
Therefore, considering the absence of systemic toxicity, pre-/post-natal and teratogenic effects observed in rat and mice by oral or pulmonary routes and the low or negligible systemic dissemination of bulk and nanoforms of CeO2, the studies reported above are considered sufficient to assess the developmental toxicity of both the bulk and nano forms of CeO2, and to conclude that no further prenatal toxicity study in rabbit is required.
REFERENCES:
- Braakhuis H. et al. Testing developmental toxicity in a second species: are the differences due to species or replication error? Regulatory Toxicology and Pharmacology 2019, 107, 104410
- Campagnolo L. & Pietroiusti A. NANoREG - Deliverable D 4.14 Prenatal toxicity study with cerium oxide and MWCNT carbon nanotubes. 2015, WP4 / Task 4.5.6, final, public version
- Davies, R. Combined repeated dose study with the reproduction/developmental toxicity screening test by oral route (gavage) in rats. CIT 2010, study report no 33179 RSR
- Davies, R. Reproduction/developmental toxicity screening test by oral route (gavage) in rats. CIT 2010, study report no 34759 RSR
- Geraerts L. et al. Tissue Distribution of Inhaled Micro- and Nano-sized Cerium Oxide Particles in Rats: Results from a 28-day Exposure Study. Toxicol Sci., 2012, 127(2): 463-73
- Gosens I. et al. Comparative hazard identification of nano- and micro-sized cerium oxide particles- based on 28-day inhalation studies in rats. Nanotoxicology, 2014, 8(6): 643-53
- He X et al. Lung deposition and extrapulmonary translocation of nano-ceria after intratracheal instillation. Nanotechnology, 2010, 21(28) :285103
- Hirst SM et al. Bio-distribution and In Vivo Antioxidant Effects of Cerium Oxide Nanoparticles in Mice. Environ Toxicol., 2011, 28(2) :107-18
- Keller J. et al. Time course of lung retention and toxicity of inhaled particles: short-term exposure to nano-Ceria. Arch Toxicol., 2014a and b, 88(11): 2033-59
- Laux P. et al. Biokinetics of nanomaterials: The role of biopersistence. NanoImpact, 2017, 6: 69–80
- Lee J. et al. Safety assessment of cerium oxide nanoparticles: combined repeated-dose toxicity with reproductive/developmental toxicity screening and biodistribution in rats. Nanotoxicology, 2020, 14 (5): 1-15
- Molina RM et al. Bioavailability, distribution and clearance of tracheally instilled, gavaged or injected cerium dioxide nanoparticles and ionic cerium. Environ. Sci.: Nano., 2014, 1: 561-573
- Moreno-Horn M. and Gebel T. Granular biodurable nanomaterials: No convincing evidence for systemic toxicity. Crit Rev Toxicol 2014, Nov; 44(10): 849-75
- NANoREG, a common European approach to the regulatory testing of nanomaterials. Final Report Part 1- Version 20170809
- Park EJ et al. Acute Toxicity and Tissue Distribution of Cerium Oxide Nanoparticles by a Single Oral Administration in Rats. Toxicol Res., 2009, 25(2): 79-84
- Schaudien D. et al. Histopathological investigation of specimens from a long term inhalation study. Report - Research F 2325, 2019
- Spézia F. Two-generation reproduction toxicity study by oral route (gavage) in rats. CIT 2011, study report no 34760 RSR
- Tentschert J. et al. Organ burden of inhaled nanoceria in a 2-year low-dose exposure study: dump or depot? Nanotoxicology 2020, 14(4): 554–576
Zirconium dioxide
An OECD 422 test has been performed according to GLP principles with the read across substance zirconium acetate, a 'water-soluble' zirconium compound.
The results of this test indicate that zirconium acetate is a substance of low toxicological concern, as the NOAEL for reproductive/developmental toxicity was considered to be higher than or equal to 1000 mg/kg bw/day (the highest dose tested, expressed as anhydrous zirconium acetate). Furthermore, litter data, pup weights and sex ratio were not affected by treatment. No clinical signs of pups were reported (Rossiello, 2013). Assuming that the more water soluble a metal compound is, the higher is its systemic bioavailability, it can be reasonably expected that the developmental toxicity potential of zirconium dioxide (an insoluble zirconium substance) after repeated oral exposure will be even of lower concern than that of zirconium acetate. Because the results on developmental toxicity obtained in an OECD 422 study should be considered as 'indicative' and should not be used on their own for covering the endpoint on developmental toxicity, an assessment was made of all other available data on zirconium dioxide and other zirconium compounds. This assessment concluded that zirconium dioxide (as well as the other zirconium compounds included in the assessment) have an extremely low potential for absorption and causing adverse effects and therefore further testing on developmental toxicity is not deemed necessary/useful. This assessment is attached to IUCLID Section 13 and summarised in the endpoint summary on toxicity to reproduction.
Reaction mass of cerium dioxide and zirconium dioxide
Based on the available information on the constituents of the reaction mass (cerium dioxide and zirconium dioxide), the performance of a second pre-natal development study with the reaction mass or either of its constituents is scientifically not considered necessary nor justified. - Reason / purpose for cross-reference:
- data waiving: supporting information
- Reason / purpose for cross-reference:
- data waiving: supporting information
- Reason / purpose for cross-reference:
- data waiving: supporting information
- Species:
- rabbit
- Endpoint:
- developmental toxicity
- Remarks:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2020
- 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:
- other: OECD 422
- Version / remarks:
- OECD 1996
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Orient Bio, Inc. (Republic of Korea)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks of age
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 1 or 2 per stainless-steel cage (255W × 465L × 200H mm3). Pregnant and lactating dams were housed individually in a poly-sulfone cage (260W × 420L × 180H mm3) with sterilized Aspen animal bedding (Bio Lab, Republic of Korea) during the study period.
- Diet (e.g. ad libitum): The sterilized commercial rodent feed (PMI Nutrition International, USA) was also provided ad libitum.
- Water (e.g. ad libitum): The water was irradiated by UV light and filtered prior to provide ad libitum.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C,
- Humidity (%): 50 ± 20%,
- Air changes (per hr): 10–20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light-dark cycle
IN-LIFE DATES: The experimental phase of this study was conducted in 2015. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- CeO2 NPs were diluted in deionized water and sonicated by the Vibra-Cell® sonifier with a 13 mm probe at 25% amplitude for 8 min. Dose formulations were mixed by a stirrer during the dosing, and dosing volume was 10 ml/kg.
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks.
- Proof of pregnancy: Mating was confirmed by the presence of sperm in the vaginal smear and/or the vaginal plug, and this was considered GD 0.
- After successful mating each pregnant female was caged (how): individually - Duration of treatment / exposure:
- - Males were administered during a 2-week premating period and during mating and up to the final sacrifice in males (total of 38 days).
- Females were administered during a 2-week premating period and during mating, gestation and up to lactation day (LD) 4(total of at least 41 days). - Frequency of treatment:
- daily
- Duration of test:
- 38 days in males and 41 days in females
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Negative control : deionized water (vehicle)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 12 males and 12 females
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dose levels were selected on the basis of the results of a preliminary study with CeO2 NPs in SD rats (5 animals/sex/group). Animals were daily dosed CeO2 NPs with 100, 300 and 1000 mg/kg dose levels for two weeks prior to mating, and dosing was continued through final sacrifice in males (total 28 days) and through gestation day (GD) 15 in females (total of at least 29days).
There was no test item-related change in all examined parameters, including clinical signs, body weight, food consumption, clinical pathology, macroscopic observation, organ weights, fertility, and cesarean section, at any doses tested. Therefore, 1000 mg/kg, which is the limit dose level, was selected as the high dose, and 300 and 100 mg/kg were determined to be the intermediate and low doses, respectively.
- Rationale for animal assignment (if not random):
Healthy animals with adequate body weight increase and exhibiting no clinical signs were used in this study. Twelve male and twelve female SD rats were divided to each of the groups to have a similar mean body weight using the Pristima system (Xybion Medical System Co., USA).
- Fasting period before blood sampling for clinical biochemistry: Yes, approximately 16 hours (overnight) prior to sacrifice - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical examinations including mortality and general clinical signs were examined twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs were examined once weekly during the study period
BODY WEIGHT: Yes
- Time schedule for examinations: Animal body weights were measured twice weekly during the premating and mating periods. Mated females were weightedon days 0, 7, 14 and 20 of gestation, and on days 0 and 4 of lactation.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured in the same days of the body weight measurement except for the mating and was calculated as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified.
OTHER:
> FOB :
Functional observations of animals, including sensory function tests (tail pinch, approach and touch response, pupillary reflex and acoustic startle response), grip strength and motor activity, were conducted with 6 animals/sex/group before necropsy (Moser 1991; Pierce and Kalivas 2007).
> HEMATOLOGY and CLINICAL CHEMISTRY :
- Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice.
- Blood for clinical pathology were collected from the caudal vena cava from 5 randomly selected animals/sex/group. Blood for hematology was placed into tubes containing potassium salt of ethylenediaminetetraacetic acid (EDTA) and then analyzed with an ADVIA2120i hematology analyzer (Siemens, Germany) for the following parameters: total red blood cell count (RBC), mean corpuscular volume (MCV), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), platelet count (PLT), reticulocyte count, total white blood cell count (WBC) and WBC differential count (absolute and relative counts of neutrophils [NEU], lymphocytes [LYM], monocytes [MON], basophils [BAS] and eosinophils [EOS]). Blood for coagulation was put into tubes containing 3.2% sodium citrate and centrifuged (approximately 3,000rpm, 10 min, at room temperature) to obtain plasma. A coagulation test was conducted with an ACL 9000 coagulation analyzer (Instrumentation Laboratory, Italy) for the following parameters: activated partial thromboplastin time (APTT) and prothrombin time (PT).
- Blood samples for clinical chemistry were placed into tubes without anticoagulant and kept at room temperature for a minimum of 90 min and then centrifuged (approximately 3000 rpm, 10 min, at room temperature) to obtain serum. Clinical chemistry analysis was conducted with a Toshiba 200 FR NEO chemistry analyzer (Toshiba Co., Japan) for the following parameters: glucose (GLU), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), alkaline phosphatase (ALP), total cholesterol (TCHO), triglyceride (TG), albumin/globulin ratio (A/G), total bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA), phospholipid (PL), creatine phosphokinase (CK), sodium (Na), inorganic phosphorus (IP), calcium (Ca), potassium (K) and chloride (Cl).
POSTMORTEM EXAMINATIONS (PARENTAL ANIMALS):
>SACRIFICE
All surviving males on the day after final dosing and females on LD 5 were humanely sacrificed with isoflurane. Blood for clinical pathology were collected from the caudal vena cava from 5 randomly selected animals/sex/group. Animals for blood collection were fasted approximately 16 hours (overnight) prior to sacrifice.
>GROSS NECROPSY
All animals were subjected to macroscopic observations.
> HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were examined and preserved in 10% neutral buffered formalin or an appropriate fixative for histopathology: ovaries, testes, uterus with cervix, brain, stomach, ileum, duodenum, jejunum, colon, cecum, rectum, liver, kidneys, adrenal glands, spinal cord (cervical, thoracic, lumbar), prostate, epididymides, seminal vesicles with coagulation glands, thyroid with parathyroid glands, trachea, lungs with bronchi, mesenteric lymph nodes, mandibular lymph nodes, urinary bladder, femur with marrow, sciatic nerve, spleen, heart, thymus and abnormal lesions. All reproductive organs and the other organs from 5 animals per sex in each group were further processed to slides and stained with hematoxylin and eosin for histopathological examinations. Kidneys were also examined in the low- and intermediate-dose groups to further investigate the treatment-related changes.
All male reproductive organs (testes, epididymides, seminal vesicles with coagulation glands and prostate) were weighed, and the following organs were weighed from 5 animals per sex in each group: liver, kidneys brain, pituitary gland, heart, thymus, spleen, ovaries, adrenal glands, lungs and uterus with cervix. Paired reproductive organs were weighed separately.
POSTMORTEM EXAMINATIONS (OFFSPRINGS):
After parturition, pup mortality and general clinical signs were examined once daily. Pup external abnormalities were recorded. Pup individual body weight and sex were recorded on post-natal day (PND) 0 and 4, and these data were reported for each litter. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Not specified
- Number of corpora lutea: Yes
- Number of implantations: Yes - Fetal examinations:
- - External examinations: Yes for external abnormalities
- Soft tissue examinations: No
- Skeletal examinations: No
- Other : number and sex of pups, stillbirths, live births, postnatal mortality, general clinical signs, presence of gross anomalies, weight gain.
- Statistics:
- Statistical analyses were conducted based on the general statistical method used in this type of toxicology study and our previous study (Lee et al. 2019). Statistical analysis was performed using the Pristima System or Statistical Analysis Systems (SAS Institute, USA), and the level of significance was taken when p < 0.05 or p < 0.01. The litter was used as a statistical unit for litter data.
Pup body weight was analyzed using one-way analysis of covariance (ANCOVA), and the litter size was used as the covariate. - Indices:
- Reproductive indices:
> Based on these mating results, the number of days the animals were confirmed to mate (precoital time) and fertility-related data, including mating, fertility, fecundity and pregnancy index, were calculated.
> The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups.
Offspring viability indices:
> After parturition, pup mortality and general clinical signs were examined once daily. Based on the parturition and pup mortality results, the delivery index (% of
dams with live pups among pregnant dams) and viability index (% of survival pups on post-natal day 4 after birth) were calculated. Pup individual body weight and sex were recorded on post-natal day (PND) 0 and 4, and these data were reported for each litter. - Historical control data:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Observations of animals during the study period did not reveal any differences in clinical examinations among the treatment and control groups.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No CeO2 NPs-related dead or moribund animals were observed during the study period.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related changes in body weight or weight gain during the study (see Figure 1 in "Attached background material")
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- In male rats of the 300 mg/kg dose group, food consumption during the pre-mating day 1–4 was significantly lower (93% of control) than in vehicle control animals but no alteration were seen afterward. No effect of the treatment was seen in the other treated groups in males and in all groups of treated females as compared to the controls animals (see Table 1 in "Any other information on results incl. tables"). This finding was considered by the authors to be incidental since it wa transient and did not have a dose response.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No overt effect of the treatment was observed as compared to the controls. In female rats of the 100 mg/kg dose group, the PT value was significantly higher (1.14-fold over control) than the respective level in the vehicle control animals. Other hematology values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 2 in "Any other information on results incl. tables"). Thus, the significantly increased PT in 100 mg/kg dose-group females was considered to be incidental since it did not have a dose-response.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No overt effect of the treatment was observed as compared to the controls. In male rats of the 1000 mg/kg dose group, the GGT value was significantly higher (2.24-fold over control) than the respective level in the vehicle control animals but the other clinical chemistry values for the CeO2 NPs-treated animals were comparable to those of the vehicle control animals (see Table 3 in "Any other information on results incl. tables"). The authors have considered the increase in GGT in 1000 mg/kg dose-group males as incidental since there were no correlated changes in organ weights and histopathological examinations.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related changes in organ weights among the treatment and control animals (see Tables 4 and 5 in "Any other information on results incl. tables").
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- In macroscopic observations, there were no treatment-related changes among the treatment and control animals.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- In male rats of the 1000 mg/kg dose group, an increased incidence of tubular basophilia in kidneys (Grade 1) was observed. No such effect were seen in females. No other effect in the examined organs were seen in the treated groups of male and females animals as compared to the controls. The increased incidence of tubular basophilia in the kidneys in 1000 mg/kg dose-group males was considered to be incidental by the autors since it also occurred sporadically in normal animals, did not have an obvious dose response and yield no correlated clinical chemistry changes. In addition, there were no toxicologically significant CeO2 NPs-related changes in other examinations for general systemic effects.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- > FOB:
In functional observations including sensory function tests (tail pinch, approach and touch response, pupillary reflex and acoustic startle response), grip strength and motor activity, there were no treatment-related changes during the study. - Number of abortions:
- no effects observed
- Description (incidence and severity):
- No aborption occured during the study.
- Pre- and post-implantation loss:
- not specified
- Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- The number of dams with live pups was equal to the number of pregnant dams (see Table 7 in "Any other information on results incl. tables").
- Early or late resorptions:
- not specified
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related changes in delivery index, viability index and perinatal death and the number of pups borns was similar in all groups (see Table 7 in "Any other information on results incl. tables").
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- No change of pregnancy duration due to treatment was observed in this study (see Tables 6 and 7 in "Any other information on results incl. tables").
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- No change in number of pregnant due to treatment was observed in this study (see Tables 6 and 7 in "Any other information on results incl. tables"). Pregnancy index was 100 in all dose-level groups.
- Other effects:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related changes in fertility results with precoital time. Mating index, Fertility index and Fecundidity index were all equal to 100 in all groups of males. Mating index, Fertility index and Pregnancy index were also found all equal to 100 whatever the dose level groups in females.
There were no treatment-related changes in reproductive (estrus cycle abnormalities, gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods (see Tables 6 and 7 in "Any other information on results incl. tables"). - Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: No systemic effect observed.
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No overt effect of the treatment was seen on pup body weight. An increase in F1 male and female pup covariate-adjusted body weights (up to 1.11-fold over control) during the post-natal period (PND 0 and 4 for males and PND 0 for females) was observed at 1000 mg/kg. (see Table 8 in "Any other information on results incl. tables"). Since there were no concurrent changes in maternal body weight, litter size or gestation length and no concurrent estrus cycle abnormalities and histopathological changes in reproductive organs, therefore, the increased pup body weight was not considered treatment-related.
- Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- No effect of the treatement was observed on the number of pup borned and on the perinatal death (see Table 7 in "Any other information on results incl. tables")
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- There was no effect of the treatment on the sex ratio (see Table 7 in "Any other information on results incl. tables").
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- There was no effect of the treatment on litter size and weight (see Tables 7 and 8 in "Any other information on results incl. tables").
- Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- There was no effect of the treatment on postnatal survival at PND4 (see Table 7 in "Any other information on results incl. tables").
- External malformations:
- no effects observed
- Description (incidence and severity):
- No pup with external abnormalities was found in this study (see Table 7 in "Any other information on results incl. tables").
- Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No peri- and post natal effect were observed.
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- In conclusion, under the experimental conditions of this study design, there were no CeO2 NPs related adverse effects in terms of general systemic signs as well as development and reproduction, at doses up to 1000 mg/kg bw/d. Therefore, the NOAEL for maternal toxicity can be set at 1000 mg/kd bw/day and the NOAEL for peri and post natal developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.
- Executive summary:
In a reproduction/developmental toxicity screening study, the effects of nano CeO2 on the general toxicity and reproductive or developmental toxicity was evaluated following daily oral administration by gavage to Sprague-Dawley rats. The study was performed according to OECD Guideline no 422 and was compliant to GLP. The study was thus considered as a key study of reliablility 1 according to Klimisch score.
Groups of 12 males and 12 females SD rats were treated by gavage with the test substance at dose levels of 0 (controls, vehicle), 100, 300 and 1000 mg/kg/day nano CeO2 in water from 2 weeks before mating, through mating and, for the females, through gestation until lactation day 4, corresponding to 38 days of treatment in males and 41 days of treatment in females. Effect of the treatment on mortality, clinical signs, body weight and body weight gain, food consumption, functional observation battery, hematology and chemical chemistry were evaluated. All animals were sacrificed at the end of the study and gross necropsy and histopathology was performed. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Precoital time and fertility-related data, including mating, fertility, fecundity, pregnancy index and delivery index were calculated. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups were evaluated. Pup mortality, viability index, individual body weight and general clinical signs were examined once daily.
Further, parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected for cerium content analysis using inductively coupled plasma mass spectrometry (ICP-MS).
No unscheduled death or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. No effect of treatment was observed in hematology and clinical biochemistry analyses and in the functional observational battery results. The treatment with the test substance induced no change in organ weight and no gross or histopathological lesion in this study.
No estrus cycle abnormalities was observed in females and no treatment-related changes in fertility results with precoital time was found. No effect of the treatment was observed on the mating index, fertility index, fecundity index and pregnancy index. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods. Pups showed no effect of treatment on survival, clinical signs, body weight and body weight gain. No pup with external abnormalities was found in this study.
Tissue distribution analysis of cerium in parental and pup tissues revealed that nano CeO2 was not detected in almost all of the samples. Only a few samples were slightly above the mean cerium content of blank samples, but it was also observed in vehicle control and there was no correlation in cerium content among the tissues and dose groups.
The authors concluded that, under the experimental conditions of this study, no nano CeO2 related adverse effects on the general systemic signs as well as on the reproductive performance or developmental toxicity was observed at doses up to 1000 mg/kg bw/day. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and therefore, the maternal toxicity can de set at 1000 mg/kg bw/ay and the NOAEL for peri and post natal developmental effects in the pups can be set at 1000 mg/kd bw/day. In addition, CeO2 NPs were not deposited in the parental or pup internal organs after repeated oral exposure.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2015
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Remarks:
- The study was performed under the NANoREG program. The data reported in this endpoint study record are those publicly available. Although the study was performed according to OECD guideline 414, the authors mentioned three main deviations from this protocol, regarding the species, the number of administered doses and the number of animals included in the experiments. The reasons for these deviations are further detailed in the section "Any other information on materials and metods incl. tables".
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- yes
- Remarks:
- There are 3 main deviations from the protocol, regarding the species, the number of administered doses and the number of animals included in the experiments (see section "Any other information on materials and methods incl. tables" for further details.
- GLP compliance:
- not specified
- Remarks:
- The study was performed under the NANoREG program. The data reported in this endpoint study record are those publicly available. The GLP status of this study is not specified in the publicly available information.
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: CD1 outbred strain (Charles River, Calco, Italy)
- Age at study initiation: 8 weeks old
- Weight at study initiation: 30 and 35 grams.
- Fasting period before study: not specified
- Housing: housed under standard laboratory conditions
- Diet (e.g. ad libitum): at libitum
- Water (e.g. ad libitum): at libitum
- Acclimation period:not specified
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 h cycle of light and dark
- remark : the authors claimed that animals were housed and mated under standard laboratory conditions
IN-LIFE DATES: From: To: Not specified - Route of administration:
- other: pulmonary exposure
- Type of inhalation exposure (if applicable):
- other: pharyngeal aspiration
- Vehicle:
- other: water/0.05% BSA
- Details on exposure:
- For inhalation exposure pharyngeal aspiration was chosen, as it is a non-invasive technique not requiring insertion of a cannula or needle in the trachea (like in intra tracheal instillation) eliminating the main source of trauma. Two doses of 5 and 20 mg/kg of body weight were used. After anaesthesia by intraperitoneal administration of 0.1 ml every 20 gr of body weight of a mixture of Rompun and Zoletil (0.1 ml and 0.750 ml, respectively, in 4.15 ml of saline solution), animals were held vertically and the tongue was gently pulled out of the mouth using forceps. Recovery from the anaesthesia usually occurred after 2 hour from the end of the procedure; survival was above 95%. Fifty microliters of CeO2 suspension (containing either 150 or 600 micrograms of CeO2 NP) were pipetted in the back of the tongue holding the nose closed and completion of two deep breaths was ascertained before tongue and nose were released. Pharyngeal aspiration was performed only once since in the preliminary experiments repeated anaesthesia was found to affect pregnancy per se.
Details of preparation of the nanoparticule supsensions were reported as follow : nanoparticles were dispersed in water/0.05% BSA and sonicated according to the Nanogenotox protocol. Briefly, 2,56 mg of nanoparticles were pre-wetted with 0.5vol% of ethanol and dispersed in1 ml sterile-filtered 0.05% w/v BSA-water. Water used for NP dispersion was obtained from a Millipore water purification system (MilliQ-water) with the following characteristics: resistivity of 18.2 MΩ-cm at 25ºC, pyrogens <0.02 EU/ml, silicates<0.1 ppb, Heavy metals ≤ 0.1 ppb, microorganisms ≤1 cfu/ml. Sonication was performed in an ice-water bath using a Watt Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a standard 13 mm disruptor horn. Nanoparticle suspensions were freshly prepared immediately before each experiment and left overs were discarded. - Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- Not specified
- Details on mating procedure:
- Pregnant females were used. No further details were specified.
- Duration of treatment / exposure:
- Once at GD 9.5
Pharyngeal aspiration was performed only once since in the preliminary experiments repeated anaesthesia was found to affect pregnancy per se. The stage of pregnancy corresponding to GD9.5 was chosen since implantation has occurred, organogenesis is still ongoing and placentation has just started. Gestational day 9.5 has been previously demonstrated to be the last stage at which placental translocation of nanoparticle can be observed after intravenous administration of NP (Yang et al. 2012). - Frequency of treatment:
- Once
- Duration of test:
- Up to the time of sacrifice : on GD 18.5.
- Dose / conc.:
- 5 mg/kg bw/day (nominal)
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 females/dose in treated groups. 10 in the control group.
- Control animals:
- yes
- Maternal examinations:
- The authors monitored the clinical situation of the animals until sacrifice (weight, behaviour, any sign of disease).
Selected maternal organs (liver, lungs, placenta) were excised for macroscopic and microscopic evaluation. Images were acquired using a Zeiss Axioplan 2 and a video camera connected to a computer. - Ovaries and uterine content:
- - At the time of sacrifice on GD 18.5, uteri from pregnant females were collected, embryos and the corresponding placentas released and analysed under a magnifier lamp and a dissection microscope for gross anatomical abnormalities.
- Number of resorptions, if present, were also recorded and classified as early or late depending on the morphology: early resorptions did not show any recognisable placental and embryonic structure, while late resorptions displayed placentas and dead embryos with evident external degenerative alterations. - Fetal examinations:
- - Embryos were weighted and the crown-rump length measured.
- After macroscopic analysis, placentas and embryos were processed for paraffin embedding following standard protocols. Sections were stained with H&E and analysed under the light microscope for any internal alteration. Images were acquired using a Zeiss Axioplan 2 and a video camera connected to a computer. - Statistics:
- Statistical analysis: Categorical variable (e.g. number of resorptions) were analysed by means of Chi-square test, or Fisher test, where appropriate; Student’s t test was used for continuous variables (e.g. crown-rump length). Mixed ANOVA was used to detect differences in the temporal pattern of changes of clinical parameters (e.g. weight gain).
- Indices:
- - Females with malformed embryos,
- n. of early resorption
- Non pregnant uteri
- Crown-rump length
- n. of total embryos - Historical control data:
- Not specified
- Clinical signs:
- not specified
- Mortality:
- not specified
- Body weight and weight changes:
- not specified
- Food consumption and compound intake (if feeding study):
- not specified
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- not specified
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- The authors found no overt toxicity in terms of miscarriage or malformations. No significant differences in the parameters analysed were observed among the experimental groups, although the average number of embryos was slightly lower in the group exposed to the highest dose of nanoceria. Once again, the difference was not statistically significant (P= 0.33, Student’s t test).
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- not specified
- Total litter losses by resorption:
- not specified
- Description (incidence and severity):
- A trend toward a higher number of resorptions and a slightly reduced number of delivered embryos was observed after pulmonary exposure to the highest dose of nanoceria. The difference was not statistically significant (P= 0.33, Student’s t test). See table of results in section Any other information on results incl. tables.
- Dead fetuses:
- not specified
- Changes in pregnancy duration:
- not specified
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- none
- Details on maternal toxic effects:
- The authors found no overt toxicity in terms of miscarriage or malformations. No significant differences in the parameters analysed were observed among the experimental groups.
- Dose descriptor:
- NOEC
- Remarks:
- pharyngeal aspiration
- Effect level:
- >= 20 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: No significant effect observed
- Remarks on result:
- other: deduced from the information in the public NANoREG report
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- not specified
- Reduction in number of live offspring:
- not specified
- Changes in sex ratio:
- not specified
- Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- The average number of embryos was slightly lower in the group exposed to the highest dose of nanoceria. The difference was not statistically significant (P= 0.33, Student’s t test).
- Changes in postnatal survival:
- not examined
- External malformations:
- no effects observed
- Description (incidence and severity):
- The authors found no overt toxicity in terms of malformations.
- Skeletal malformations:
- not specified
- Visceral malformations:
- not specified
- Other effects:
- no effects observed
- Description (incidence and severity):
- There were no effect of treatment on crown-rump lenght of the pups (see table of results in Any information on result incl. tables).
- Dose descriptor:
- NOAEC
- Remarks:
- pharyngeal aspiration
- Effect level:
- >= 20 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- other: No significant effect observed
- Remarks on result:
- other: deduced from the information in the public NANoREG report
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- The authors concluded that their data suggest a lack of serious embryo toxicity after pulmonary exposure to cerium dioxide.
- Executive summary:
In the prenatal toxicity study, performed with a method equivalent to OECD 414, pregnant CD-1 mice have been exposed at gestational day 9.5 to 0 (controls), 5 and 20 mg/kg bw nano cerium oxide, dispersed in a water/0.05% BSA solution, through pulmonary route using a single pharyngeal aspiration. The clinical situation of the animals (weight, behaviour, any sign of disease) was monitored until sacrifice on gestational day 18.5. Selected maternal organs (liver, lungs, placenta) were excised for macroscopic evaluation and uteri from pregnant females were collected, embryos and the corresponding placentas released and analysed for gross anatomical abnormalities. Number of resorptions, if present, were also recorded and classified as early or late depending on the morphology. Embryos were weighted and the crown-rump length measured. After macroscopic analyses, placentas and embryos were processed for paraffin embedding following standard protocols for microscopic analyses.
The authors found no overt toxicity in terms of miscarriage or malformations. No significant differences in the analysed parameters were observed among the experimental groups. A trend toward a higher number of resorption and a slightly reduced number of delivered embryos were observed with the highest dose of cerium dioxide but the difference was not statistically significant. There were no effect of treatment on crown-rump lenght of the pups.
The authors concluded that their data suggest a lack of serious embryo toxicity after pulmonary exposure to cerium dioxide.
From the perspective of regulators and policy makers, the authors concluded that their data imply that unintended pulmonary exposure to cerium oxide nanoparticles at doses which can be realistically expected in occupational and environmental settings should not pose peculiar risk to pregnant women.
The robust study summary was build with the available information for public from the NANoREG Deliverable 4.14 Prenatal toxicity study with cerium oxide and MWCNT carbon nanotubes.
Referenceopen allclose all
> Tissue distribution of cerium:
Tissue distribution analysis of cerium in parental and pup tissues revealed that CeO2 NPs were not detected in almost all of the samples. Only a few samples were slightly above the mean cerium content of blank samples, but it was also observed in vehicle control and there was no correlation in cerium content among the tissues and dose groups.
> Tables:
Table 1: Food consumption of CeO2 NPs-treated males and females during the study period.
CeO2 NPs (mg/kg bw/day) |
0 |
100 |
300 |
1000 |
Males |
||||
Pre-mating day 1–4 (g) |
30.6 ± 1.9 |
29.3 ± 0.8 |
28.6 ± 1.5* |
30.5 ± 1.6 |
Pre-mating day 4–8 (g) |
31.3 ± 2.2 |
30.4 ± 1.2 |
30.2 ± 1.6 |
31.2 ± 1.4 |
Pre-mating day 8–11 (g) |
31.4 ± 2.3 |
31.5 ± 1.5 |
30.5 ± 2.3 |
32.2 ± 1.2 |
Pre-mating day 11–14 (g) |
32.3 ± 2.0 |
32.6 ± 1.8 |
31.4 ± 2.4 |
32.7 ± 1.1 |
Total period (g, pre-mating day 1–14) |
31.4 ± 2.0 |
30.9 ± 1.1 |
30.2 ± 1.9 |
31.7 ± 1.0 |
Females |
||||
Pre-mating day 1–4 (g) |
21.1 ± 1.6 |
20.2 ± 1.1 |
21.3 ± 1.1 |
21.2 ± 1.6 |
Pre-mating day 4–8 (g) |
21.8 ± 1.2 |
21.4 ± 0.8 |
22.7 ± 1.2 |
22.2 ± 1.6 |
Pre-mating day 8–11 (g) |
21.9 ± 1.7 |
22.3 ± 1.4 |
23.0 ± 2.2 |
22.1 ± 1.9 |
Pre-mating day 11–14 (g) |
23.0 ± 1.5 |
23.0 ± 1.5 |
23.7 ± 0.8 |
23.3 ± 1.9 |
Gestation day 0–7 (g) |
25.6 ± 1.7 |
26.0 ± 2.6 |
25.7 ± 2.2 |
26.8 ± 1.4 |
Gestation day 7–14 (g) |
25.5 ± 1.5 |
25.9 ± 2.3 |
25.9 ± 2.3 |
25.8 ± 2.0 |
Gestation day 14–20 (g) |
29.8 ± 2.1 |
29.2 ± 1.9 |
29.3 ± 1.9 |
31.5 ± 2.0 |
Post-natal day 0–4 (g) |
35.8 ± 5.0 |
36.8 ± 3.2 |
35.9 ± 6.1 |
40.1 ± 4.1 |
Total period (g, pre-mating day 1 to lactation day 4) |
25.0 ± 1.1 |
25.0 ± 1.4 |
25.4 ± 1.2 |
25.9 ± 1.3 |
*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=12, mean ± SD).
Table 2: Hematology results of CeO2 NPs-treated males and females during the study period.
|
Males |
Females |
||||||
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
0 |
100 |
300 |
1000 |
RBC (106/µL) |
9.1 ± 0.4 |
9.1 ± 0.3 |
8.9 ± 0.5 |
8.9 ± 0.1 |
7.8 ± 0.5 |
7.7 ± 0.3 |
7.9 ± 0.5 |
8.1 ± 0.2 |
HGB (g/dL) |
16.6 ± 0.6 |
16.9 ± 0.4 |
16.8 ± 0.6 |
16.8 ± 0.6 |
14.9 ± 0.9 |
14.8 ± 0.3 |
14.8 ± 0.9 |
15.3 ± 0.5 |
HCT (%) |
51.0 ± 2.5 |
51.4 ± 2.0 |
50.7 ± 2.1 |
50.4 ± 2.1 |
46.0 ± 2.6 |
45.6 ± 1.4 |
45.5 ± 2.7 |
47.0 ± 1.5 |
MCV (fL |
56.1 ± 1.7 |
56.2 ± 0.9 |
57.2 ± 1.8 |
56.9 ± 1.9 |
59.4 ± 0.9 |
59.4 ± 1.2 |
57.9 ± 2.0 |
58.2 ± 1.2 |
MCH (pg) |
18.3 ± 0.4 |
18.5 ± 0.3 |
18.9 ± 0.7 |
18.9 ± 0.5 |
19.3 ± 0.2 |
19.3 ± 0.5 |
18.8 ± 0.7 |
18.9 ± 0.3 |
MCHC (g/dL) |
32.5 ± 0.4 |
33.0 ± 0.7 |
33.1 ± 0.6 |
33.3 ± 0.4 |
32.5 ± 0.5 |
32.5 ± 0.5 |
32.5 ± 0.4 |
32.4 ± 0.4 |
PLT (103/µL) |
1130.8 ± 149.7 |
1024.2 ± 133.7 |
940.4 ± 41.3 |
1101.6 ± 84.8 |
1351.0 ± 167.3 |
1213.2 ± 174.2 |
1258.4 ± 222.7 |
1305.0 ± 253.0 |
RET (%) |
2.5 ± 0.5 |
2.5 ± 0.3 |
2.5 ± 0.3 |
2.5 ± 0.6 |
5.8 ± 1.3 |
6.4 ± 1.7 |
6.3 ± 1.4 |
6.2 ± 1.7 |
RETA (109/µL |
223.8 ± 41.4 |
226.0 ± 22.2 |
223.8 ± 31.6 |
218.4 ± 48.7 |
448.3 ± 78.3 |
492.2 ± 142.9 |
489.7 ± 77.8 |
502.9 ± 136.4 |
WBC(103/µL) |
10.6 ± 3.3 |
11.6 ± 4.1 |
12.4 ± 1.0 |
11.0 ± 2.2 |
9.5 ± 2.5 |
9.8 ± 2.4 |
11.2 ± 1.1 |
12.8 ± 3.2 |
NEU (%) |
14.8 ± 6.9 |
11.4 ± 1.7 |
12.1 ± 3.0 |
13.4 ± 2.5 |
12.2 ± 5.0 |
13.3 ± 2.2 |
12.8 ± 3.2 |
13.2 ± 2.8 |
NEUA(103/µL) |
1.5 ± 0.7 |
1.4 ± 0.7 |
1.5 ± 0.5 |
1.5 ± 0.5 |
1.2 ± 0.7 |
1.3 ± 0.3 |
1.4 ± 0.2 |
1.7 ± 0.8 |
LYM (%) |
81.3 ± 6.7 |
84.7 ± 2.1 |
83.2 ± 3.6 |
81.7 ± 2.0 |
81.5 ± 5.2 |
80.5 ± 2.0 |
80.7 ± 4.0 |
80.3 ± 3.2 |
LYMA(103/µL) |
8.7 ± 2.8 |
9.8 ± 3.3 |
10.3 ± 0.7 |
9.0 ± 1.7 |
7.7 ± 2.0 |
7.9 ± 2.1 |
9.1 ± 1.4 |
10.2 ± 2.3 |
EOS (%) |
0.8 ± 0.2
|
0.9 ± 0.2 |
0.9 ± 0.3 |
1.0 ± 0.4 |
0.8 ± 0.4 |
0.6 ± 0.2 |
0.7 ± 0.3 |
0.7 ± 0.3 |
EOSA (103/µL) |
0.09 ± 0.05 |
0.10 ± 0.03 |
0.12 ± 0.05 |
0.11 ± 0.04 |
0.07 ± 0.02 |
0.06 ± 0.04 |
0.07 ± 0.02 |
0.09 ± 0.04 |
MON (%) |
1.9 ± 0.6 |
1.9 ± 0.6 |
2.3 ± 0.7 |
2.5 ± 0.6 |
4.3 ± 0.7 |
4.0 ± 1.2 |
4.6 ± 0.9 |
4.6 ± 0.8 |
MONA (103/µL) |
0.2 ± 0.1 |
0.3 ± 0.2 |
0.3 ± 0.1 |
0.3 ± 0.0 |
0.4 ± 0.1 |
0.4 ± 0.1 |
0.5 ± 0.1 |
0.6 ± 0.2 |
BAS (%) |
0.6 ± 0.1 |
0.5 ± 0.1 |
0.7 ± 0.1 |
0.6 ± 0.2 |
0.5 ± 0.1 |
0.5 ± 0.2 |
0.4 ± 0.1 |
0.4 ± 0.1 |
BASA (103/µL) |
0.06 ± 0.02 |
0.06 ± 0.03 |
0.09 ± 0.01 |
0.06 ± 0.01 |
0.04 ± 0.01 |
0.05 ± 0.03 |
0.05 ± 0.01 |
0.05 ± 0.02 |
LUC (%) |
0.7 ± 0.3 |
0.6 ± 0.1 |
0.7 ± 0.1 |
0.8 ± 0.1 |
0.8 ± 0.3 |
1.1 ± 0.1 |
0.8 ± 0.3 |
0.8 ± 0.2 |
LUCA (103/µL) |
0.07 ± 0.05 |
0.07 ± 0.04 |
0.09 ± 0.02 |
0.09 ± 0.01 |
0.07 ± 0.01 |
0.10 ± 0.03 |
0.09 ± 0.03 |
0.10 ± 0.03 |
PT (sec) |
14.0 ± 1.7 |
13.7 ± 0.8 |
16.0 ± 2.7 |
13.6 ± 0.7 |
13.6 ± 1.2 |
15.5 ± 0.3* |
15.3 ± 0.9 |
14.4 ± 1.5 |
APTT (sec) |
17.5 ± 0.7 |
18.0 ± 0.8 |
18.3 ± 0.9 |
17.8 ± 1.0
|
6.1 ± 0.7 |
15.6 ± 1.2 |
14.8 ± 1.0 |
15.0 ± 0.9 |
*: Represent a significant difference at the p<0.05 level compared to the vehicle control (n=5, mean ± SD).
Table 3: Clinical chemistry results of CeO2 NPs-treated males and females during the study period.
|
Males |
Females |
||||||
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
0 |
100 |
300 |
1000 |
GLU (mg/dL) |
158.9 ± 20.3 |
149.2 ± 28.8 |
157.6 ± 45.1 |
148.5 ± 29.1 |
131.2 ± 22.4 |
116.0 ± 15.1 |
108.8 ± 10.7 |
139.1 ± 17.1 |
BUN (mg/dL) |
15.1 ± 1.6 |
13.7 ± 1.5 |
14.3 ± 1.7 |
13.6 ± 1.8 |
24.1 ± 4.7 |
19.4 ± 3.4 |
21.2 ± 4.1 |
20.3 ± 3.6 |
CREA (mg/dL) |
0.49 ± 0.04 |
0.48 ± 0.03 |
0.47 ± 0.04 |
0.47 ± 0.06 |
0.57 ± 0.08 |
0.51 ± 0.03 |
0.54 ± 0.04 |
0.56 ± 0.03 |
TP (g/dL) |
6.6 ± 0.3 |
6.7 ± 0.2 |
6.6 ± 0.2 |
6.6 ± 0.4 |
7.0 ±0.4 |
7.0 ± 0.4
|
7.0 ± 0.2 |
7.1 ± 0.3 |
ALB (g/dL) |
4.3 ± 0.2 |
4.2 ± 0.1 |
4.2 ± 0.1 |
4.2 ± 0.2 |
4.5 ±0.2 |
4.6 ± 0.3 |
4.5 ± 0.1 |
4.6 ± 0.1 |
A/G (ratio) |
1.8 ± 0.2 |
1.7 ± 0.1 |
1.8 ± 0.1 |
1.8 ± 0.1 |
1.8 ±0.1 |
2.0 ± 0.2 |
1.9 ± 0.1 |
1.9 ± 0.1 |
AST (IU/L) |
149.4 ± 21.9 |
137.5 ± 34.5 |
129.9 ± 4.3 |
153.9 ± 18.5 |
142.7 ± 44.3 |
129.1 ± 23.1 |
135.6 ± 17.7 |
121.3 ± 8.0 |
ALT (IU/L) |
33.4 ± 5.7 |
28.6 ± 5.6 |
32.5 ± 3.0 |
31.5 ± 3.9 |
40.2 ± 8.0 |
41.5 ± 8.1 |
45.1 ± 5.2 |
39.8 ± 2.6 |
TBIL (mg/dL) |
0.14 ± 0.02 |
0.12 ± 0.01 |
0.15 ± 0.02 |
0.13 ± 0.02 |
0.13 ± 0.02 |
0.12 ± 0.02 |
0.13 ± 0.02 |
0.12 ± 0.02 |
GGT (IU/L) |
0.41 ± 0.23 |
0.65 ± 0.13 |
0.60 ± 0.12 |
0.92 ± 0.18** |
0.59 ± 0.22 |
0.78 ± 0.10 |
0.67 ± 0.27 |
0.71 ± 0.38 |
ALP (IU/L) |
581.6 ± 179.2 |
510.3 ± 110.0 |
467.4 ± 57.3 |
514.4 ± 66.4 |
332.3 ± 111.0 |
293.1 ± 58.0 |
232.7 ± 31.7 |
267.1 ± 83.9 |
TCHO (mg/dL) |
70.8 ± 26.9 |
64.2 ± 19.5 |
60.0 ± 8.9 |
68.4 ± 10.2 |
49.8 ± 16.0 |
58.0 ± 16.5 |
53.8 ± 10.8 |
55.2 ± 12.8 |
TG (mg/dL) |
31.8 ± 9.3 |
34.2 ± 11.8 |
23.9 ± 10.0 |
21.4 ± 10.9 |
38.9 ± 22.5 |
36.9 ± 13.1 |
41.7 ± 14.9 |
54.7 ± 25.5 |
Ca (mg/dL) |
11.1 ± 0.3 |
11.3 ± 0.4 |
11.2 ± 0.2 |
11.0 ± 0.7 |
11.7 ± 0.4 |
11.8 ± 0.5 |
11.7 ± 0.3 |
11.9 ± 0.2 |
IP (mg/dL) |
10.2 ± 0.5 |
10.3 ± 0.6 |
10.5 ± 0.4 |
10.3 ± 0.7 |
9.9 ± 0.8 |
10.2 ± 0.1 |
10.3 ± 1.2 |
9.8 ± 0.7 |
K (mmol/L) |
8.3 ± 0.9 |
7.5 ± 0.9 |
8.4 ± 0.9 |
7.8 ± 1.6 |
8.6 ± 0.5 |
8.3 ± 0.5 |
8.2 ± 1.1 |
8.2 ± 1.7 |
CK (IU/L) |
715.6 ± 197.6 |
584.2 ± 203.2 |
483.6 ± 79.4 |
652.0 ± 218.0 |
611.8 ± 331.8 |
518.0 ± 112.9 |
570.6 ± 129.1 |
448.8 ± 130.3 |
PL (mg/dL) |
102.4 ± 25.0 |
93.6 ± 22.7 |
92.0 ± 7.3 |
99.2 ± 10.4 |
106.8 ± 26.9 |
116.8 ± 24.8 |
108.6 ± 14.8 |
115.8 ± 19.1 |
Na (mmol/L) |
148.0 ± 0.7 |
147.8 ± 1.9 |
147.4 ± 1.1 |
147.8 ± 1.6 |
144.8 ± 1.3 |
145.0 ± 1.9 |
145.0 ± 1.4 |
146.0 ± 1.6 |
Cl (mmol/L) |
102.4 ± 1.7 |
101.6 ± 0.6 |
102.6 ± 1.3 |
102.4 ± 0.9 |
101.4 ± 0.6 |
101.2 ± 1.5 |
101.6 ± 1.1 |
102.2 ± 2.4 |
**: Represent a significant difference at the p<0.01 level compared to the vehicle control (n=5, mean ± SD).
Table 4: Absolute and relative organ weights of CeO2 NPs-treated males during the study period.
CeO2 NPs (mg/kg) |
|
0 |
100 |
300 |
1000 |
Terminal body weighta(g) |
(g) |
427.0 ± 38.7 |
439.2 ± 23.7 |
436.4 ± 36.0 |
448.9 ± 28.6 |
N |
12 |
12 |
12 |
12 |
|
Adrenal glands |
Absolute (g) |
0.06 ± 0.01 |
0.07 ± 0.01 |
0.06 ± 0.01 |
0.07 ± 0.00 |
Relativeb(%) |
0.016 ± 0.003 |
0.016 ± 0.002 |
0.016 ± 0.002 |
0.015 ± 0.001 |
|
N |
5 |
5 |
5 |
5 |
|
Brain |
Absolute (g) |
1.97 ± 0.12 |
1.99 ± 0.07 |
2.03 ± 0.09 |
2.00 ± 0.12 |
Relative (%) |
0.484 ± 0.068 |
0.460 ± 0.023 |
0.497 ± 0.030 |
0.461 ± 0.013 |
|
N |
5 |
5 |
5 |
5 |
|
Heart |
Absolute (g) |
1.25 ± 0.12 |
1.29 ± 0.14 |
1.33 ± 0.13 |
1.32 ± 0.11 |
Relative (%) |
0.304 ± 0.023 |
0.300 ± 0.030 |
0.325 ± 0.024 |
0.303 ± 0.028 |
|
N |
5 |
5 |
5 |
5 |
|
Kidneys |
Absolute (g) |
3.25 ± 0.19 |
3.40 ± 0.15 |
3.50 ± 0.33 |
3.47 ± 0.42 |
Relative (%) |
0.794 ± 0.071 |
0.788 ± 0.050 |
0.854 ± 0.047 |
0.797 ± 0.070 |
|
N |
5 |
5 |
5 |
5 |
|
Liver |
Absolute (g) |
11.71 ± 2.07 |
12.74 ± 0.71 |
12.12 ± 2.09 |
12.51 ± 1.41 |
Relative (%) |
2.825 ± 0.217 |
2.950 ± 0.1063 |
2.940 ± 0.246 |
2.870 ± 0.197 |
|
N |
5 |
5 |
5 |
5 |
|
Pituitary gland |
Absolute (g) |
0.01 ± 0.00 |
0.01 ± 0.00 |
0.01 ± 0.00 |
0.01 ± 0.00 |
Relative (%) |
0.003 ± 0.000 |
0.003 ± 0.000 |
0.003 ± 0.000 |
0.003 ± 0.000 |
|
N |
5 |
5 |
5 |
5 |
|
Prostate |
Absolute (g) |
0.62 ± 0.12 |
0.59 ± 0.17 |
0.66 ± 0.13 |
0.65 ± 0.09 |
Relative (%) |
0.144 ± 0.026 |
0.135 ± 0.041 |
0.151 ± 0.031 |
0.146 ± 0.018 |
|
N |
12 |
12 |
12 |
12 |
|
Spleen |
Absolute (g) |
0.66 ± 0.13 |
0.67 ± 0.15 |
0.65 ± 0.13 |
0.69 ± 0.08 |
Relative (%) |
0.158 ± 0.013 |
0.155 ± 0.033 |
0.158 ± 0.020 |
0.158 ± 0.013 |
|
N |
5 |
5 |
5 |
5 |
|
Thymus |
Absolute (g) |
0.34 ± 0.02 |
0.39 ± 0.08 |
0.35 ± 0.11 |
0.32 ± 0.06 |
Relative (%) |
0.083 ± 0.010 |
0.090 ± 0.018 |
0.084 ± 0.019 |
0.072 ± 0.011 |
|
N |
5 |
5 |
5 |
5 |
|
Lungs |
Absolute (g) |
1.50 ± 0.13 |
1.58 ± 0.12 |
1.57 ± 0.17 |
1.62 ± 0.07 |
Relative (%) |
0.367 ± 0.041 |
0.367 ± 0.031 |
0.383 ± 0.021 |
0.374 ± 0.021 |
|
N |
5 |
5 |
5 |
5 |
|
Right testis |
Absolute (g) |
1.70 ± 0.17 |
1.74 ± 0.13 |
1.69 ± 0.11 |
1.65 ± 0.12 |
Relative (%) |
0.400 ± 0.049 |
0.396 ± 0.031 |
0.391 ± 0.051 |
0.369 ± 0.028 |
|
N |
12 |
12 |
12 |
12 |
|
Left testis |
Absolute (g) |
1.73 ± 0.16 |
1.74 ± 0.12 |
1.71 ± 0.11 |
1.67 ± 0.12 |
Relative (%) |
0.407 ± 0.051 |
0.397 ± 0.026 |
0.394 ± 0.044 |
0.372 ± 0.030 |
|
N |
12 |
12 |
12 |
12 |
|
Right epididymis |
Absolute (g) |
0.66 ± 0.06 |
0.69 ± 0.06 |
0.68 ± 0.06 |
0.66 ± 0.07 |
Relative (%) |
0.155 ± 0.017 |
0.158 ± 0.016 |
0.156 ± 0.021 |
0.148 ± 0.021 |
|
N |
12 |
12 |
12 |
12 |
|
Left epididymis |
Absolute (g) |
0.67 ± 0.07 |
0.68 ± 0.06 |
0.66 ± 0.06 |
0.65 ± 0.06 |
Relative (%) |
0.157 ± 0.019 |
0.156 ± 0.014 |
0.152 ± 0.019 |
0.145 ± 0.018 |
|
N |
12 |
12 |
12 |
12 |
|
Seminal vesicles with coagulating glands |
Absolute (g) |
1.68 ± 0.21 |
1.65 ± 0.29 |
1.68 ± 0.22 |
1.69 ± 0.21 |
Relative (%) |
0.396 ± 0.047 |
0.378 ± 0.072 |
0.388 ± 0.057 |
0.377 ± 0.048 |
|
N |
12 |
12 |
12 |
12 |
N=5 or 12, mean ± SD.
a: Terminal body weight were measured immediately before necropsy.
b: Organ weight/terminal body weight ratio.
Table 5: Absolute and relative organ weights of CeO2 NPs-treated females during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
|
Terminal body weight |
(g) |
314.5 ± 19.9 |
316.7 ± 24.1 |
311.0 ± 23.7 |
316.8 ± 19.8 |
Adrenal glands |
Absolute (g) |
0.08 ± 0.01 |
0.07 ± 0.01 |
0.08 ± 0.01 |
0.08 ± 0.01 |
Relative (%) |
0.028 ± 0.003 |
0.024 ± 0.003 |
0.027 ± 0.003 |
0.027 ± 0.002 |
|
Brain |
Absolute (g) |
1.94 ± 0.09 |
1.93 ± 0.08 |
2.02 ± 0.08 |
1.96 ± 0.05 |
Relative (%) |
0.652 ± 0.036 |
0.650 ± 0.022 |
0.692 ± 0.028 |
0.654 ± 0.030 |
|
Heart |
Absolute (g) |
1.00 ± 0.06 |
0.95 ± 0.04 |
0.97 ± 0.07 |
1.04 ± 0.06 |
Relative (%) |
0.334 ± 0.013 |
0.321 ± 0.017 |
0.334 ± 0.026 |
0.346 ± 0.019 |
|
Kidneys |
Absolute (g) |
2.28 ± 0.19 |
2.23 ± 0.21 |
2.14 ± 0.14 |
2.23 ± 0.07 |
Relative (%) |
0.763 ± 0.028 |
0.750 ± 0.051 |
0.734 ± 0.036 |
0.744 ± 0.027 |
|
Liver |
Absolute (g) |
10.52 ± 1.21 |
10.40 ± 0.87 |
10.19 ± 0.30 |
10.91 ± 0.73 |
Relative (%) |
3.522 ± 0.266 |
3.503 ± 0.277 |
3.495 ± 0.169 |
3.638 ± 0.259 |
|
Pituitary gland |
Absolute (g) |
0.02 ± 0.00 |
0.02 ± 0.00 |
0.02 ± 0.00 |
0.02 ± 0.00 |
Relative (%) |
0.006 ± 0.001 |
0.006 ± 0.001 |
0.006 ± 0.001 |
0.006 ± 0.000 |
|
Spleen |
Absolute (g) |
0.62 ± 0.07 |
0.69 ± 0.09 |
0.65 ± 0.08 |
0.70 ± 0.12 |
Relative (%) |
0.209 ± 0.021 |
0.231 ± 0.029 |
0.223 ± 0.032 |
0.232 ± 0.043 |
|
Thymus |
Absolute (g) |
0.31 ± 0.03 |
0.31 ± 0.05 |
0.31 ± 0.08 |
0.38 ± 0.12 |
Relative (%) |
0.104 ± 0.013 |
0.104 ± 0.021 |
0.105 ± 0.027 |
0.125 ± 0.041 |
|
Lungs |
Absolute (g) |
1.34 ± 0.04 |
1.29 ± 0.06 |
1.42 ± 0.05 |
1.36 ± 0.11 |
Relative (%) |
0.451 ± 0.013 |
0.435 ± 0.018 |
0.486 ± 0.022 |
0.453 ± 0.034 |
|
Uterus |
Absolute (g) |
0.78 ± 0.12 |
0.78 ± 0.09 |
0.83 ± 0.12 |
0.73 ± 0.06 |
Relative (%) |
0.260 ± 0.039 |
0.262 ± 0.030 |
0.284 ± 0.035 |
0.243 ± 0.026 |
|
Right ovary |
Absolute (g) |
0.07 ± 0.01 |
0.06 ± 0.01 |
0.06 ± 0.01 |
0.06 ± 0.01 |
Relative (%) |
0.021 ± 0.002 |
0.021 ± 0.004 |
0.019 ± 0.003 |
0.019 ± 0.004 |
|
Left Ovary |
Absolute (g) |
0.07 ± 0.02 |
0.06 ± 0.01 |
0.06 ± 0.01 |
0.05 ± 0.01 |
Relative (%) |
0.023 ± 0.006 |
0.020 ± 0.002 |
0.020 ± 0.005 |
0.018 ± 0.002 |
|
N=5, mean ± SD.
Table 6: Fertility with precoital time results of CeO2 NPs treated males during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
Males |
||||
Mating indexa |
100 |
100 |
100 |
100 |
Fertility indexb |
100 |
100 |
100 |
100 |
Fecundity indexc |
100 |
100 |
100 |
100 |
Females |
||||
Mating indexd |
100 |
100 |
100 |
100 |
Fertility indexe |
100 |
100 |
100 |
100 |
Pregnancy indexf |
100 |
100 |
100 |
100 |
Precoital Time (day) |
3.3 ± 3.6 |
1.8 ± 0.6 |
2.2 ± 1.1 |
1.9 ± 1.1 |
N=12, mean ± SD.
a: (No. of males with evidence of mating/No. of males paired) x 100.
b: (No. of males impregnating a female/No. of males paired) x 100.
c: (No. of males impregnating a female/No. of males with evidence of mating) x 100.
d: (No. of females with evidence of mating/No. of females paired) x 100.
e: (No. of pregnant females/No. of females paired) x 100.
f: (No. of pregnant females/No. of females with evidence of mating) x100.
Table 7: Reproductive and litter findings results of CeO2 NPs-treated females during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
Gestation period (day) |
21.5 ± 0.4 |
21.5 ± 0.3 |
21.6 ± 0.3 |
21.8 ± 0.4 |
Corpora lutea (N) |
17.8 ± 2.8 |
16.5 ± 2.6 |
16.3 ± 1.7 |
17.4 ± 2.0 |
Implantations (N) |
15.3 ± 3.1 |
15.3 ± 2.4 |
14.8 ± 2.1 |
15.4 ± 2.0 |
Pups born (N) |
14.8 ± 3.1 |
14.4 ± 2.4 |
13.9 ± 2.0 |
14.8 ± 2.1 |
Perinatal death (N) |
0.08 ± 0.29 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.25 ± 0.45 |
Unaccounted-for sitesa(%) |
2.8 ± 4.5 |
5.5 ± 5.8 |
6.2 ± 4.5 |
3.9 ± 4.5 |
Sex ratiob(%) |
106.9 ± 62.6 |
93.9 ± 28.2 |
105.3 ± 55.5 |
130.7 ± 66.8 |
Live litter size (N) PND 0 PND 4 |
14.8 ± 3.1 14.8 ± 3.1 |
14.4 ± 2.4 14.3 ± 2.4 |
13.9 ± 2.0 13.8 ± 2.0 |
14.6 ± 2.1 14.4 ± 2.0 |
Viability indexc(%) |
100.0 |
99.4 ± 1.9 |
98.9 ± 2.6 |
99.0 ± 2.4 |
Delivery indexd(%) |
100.0 |
100.0 |
100.0 |
100.0 |
Pups with external abnormalities |
0 |
0 |
0 |
0 |
N = 12, mean ± SD.
a: (No. of implantation sites/litter) - (No. of live pups at birth/litter)/No. of implantation sites/litter x 100.
b: (No. of male pups on PND 0/litter)/(No. of female pups on PND 0/litter) x 100.
c: (No. of live pups on PND 4/litter)/(No. of live pups at birth/litter) x 100.
d: (No. of dams with live pups)/(No. of pregnant dams) x 100.
Table 8: F1 pups body weights of CeO2 NPs-treated parental animals during the study period.
CeO2 NPs (mg/kg) |
0 |
100 |
300 |
1000 |
F1 male pups |
||||
PND 0 |
||||
Body weight (g) |
6.5 ± 0.4 |
6.7 ± 0.3 |
6.8 ± 0.6 |
7.0 ± 0.5 |
Covariate-adjusted mean (g) |
6.5 |
6.7 |
6.8 |
7.0* |
PND 4 |
||||
Body weight (g) |
10.1 ± 0.8 |
10.7 ± 1.1 |
11.0 ± 1.1 |
11.0 ± 1.2 |
Covariate-adjusted mean (g) |
10.1 |
10.6 |
10.9 |
11.2* |
F1 female pups |
||||
PND 0 |
||||
Body weight (g) |
6.2 ± 0.4 |
6.3 ± 0.3 |
6.4 ± 0.6 |
6.7 ± 0.6 |
Covariate-adjusted mean (g) |
6.2 |
6.3 |
6.4 |
6.7* |
PND 4 |
||||
Body weight (g) |
9.5 ± 0.9 |
10.0 ± 1.0 |
10.4 ± 1.2 |
10.6 ± 1.4 |
Covariate-adjusted mean (g) |
9.6 |
10.1 |
10.4 |
10.5 |
N =12, mean ± SD.
*Represent a significant difference at the p<0.05 level compared to the vehicle control.
|
n. of mice |
Females with malformed embryos |
n. of early resorptions
|
Non pregnant uteri |
Crown-rump-length |
n. of total embryos |
CTRL |
10 |
0 |
0 |
0 |
16.8 mm |
170/10 = 17 |
CeO2 JRCNM02102a 5 mg/kg |
5 |
0 |
1 |
0 |
17.1 mm |
88/5 = 17.6 |
CeO2 JRCNM02102a 20 mg/kg |
5 |
0 |
2 |
0 |
16.9 mm |
72/5 = 14.4 |
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no adverse effect observed
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
No study is available for the reaction mass of cerium dioxide and zirconium dioxide. Therefore, a conclusion on the endpoint was reached considering information on its constituents, zirconium dioxide and cerium dioxide.
Cerium dioxide
The pre- and post-natal developmental toxicity and the potential for teratogenicity of nano cerium dioxide was assessed in two studies.
The first one is an OECD 422 study performed in compliance with GLP that evaluated the effects of a polyhedral nano cerium dioxide with a mean particle size of about 14 nm on the general toxicity, the reproductive and peri- and post-natal developmental toxicity in Sprague-Dawley rats following daily oral administration using gavage (Lee et al., 2020). Further, in this study, the authors have measured the cerium content in parental animal tissues and tissues of pups.
In the second study, performed according to a method similar to OECD guideline 414, pregnant CD-1 mice received nano cerium dioxide (JRCNM01202a), generally described as globular with a primary particle size of 40 nm, via a single pharyngeal aspiration (pulmonary exposure) and effects of the treatment was observed in the dams and in the pups (Campagnolo and Pietroiusti, 2015).
Both studies are summarised below:
In a reproduction/developmental toxicity screening study, the effects of nano cerium dioxide on the general toxicity and reproductive or developmental toxicity was evaluated following daily oral administration by gavage to Sprague-Dawley rats. The study was performed according to OECD guideline 422 and was compliant to GLP. The study was thus considered as a key study of reliability 1 according to the Klimisch scoring system.
Groups of 12 male and 12 female Sprague-Dawley rats were treated by gavage with the test substance at dose levels of 0 (controls, vehicle), 100, 300 and 1000 mg/kg bw/day nano cerium dioxide in water from 2 weeks before mating, through mating and, for the females, through gestation until lactation day 4, corresponding to 38 days of treatment in males and 41 days of treatment in females. Effects of the treatment on mortality, clinical signs, body weight and body weight gain, food consumption, functional observation battery, hematology and chemical chemistry were evaluated. All animals were sacrificed at the end of the study and gross necropsy and histopathology was performed. The progress and completion of parturition was monitored twice daily, including signs of parturition, premature delivery, abortion, and prolonged or difficult parturition. Precoital time and fertility-related data, including mating, fertility, fecundity, pregnancy index and delivery index were calculated. Pregnant females were allowed to access their litters, and then the gestation duration, number of dead and live pups, runts, sexing of live pups were evaluated. Pup mortality, viability index, individual body weight and general clinical signs were examined once daily. Further, parental animal tissues (blood, liver, lungs and kidneys) and pup tissues (blood, liver, lungs and kidneys) were collected for cerium content analysis using inductively coupled plasma mass spectrometry (ICP-MS).
No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. No effect of treatment was observed in hematology and clinical biochemistry analyses and in the functional observational battery results. The treatment with the test substance induced no change in organ weight and no gross or histopathological lesions in this study.
No estrus cycle abnormalities was observed in females and no treatment-related changes in fertility results with precoital time were found. No effect of the treatment was observed on the mating index, fertility index, fecundity index and pregnancy index. There were no treatment-related changes in reproductive (gestation period, corpora lutea, implantation sites, pups born, perinatal death, delivery index and sex ratio) and litter finding (live litter size, viability index) parameters during the gestation and lactation periods. Pups showed no effect of treatment on survival, clinical signs, body weight and body weight gain.
No pup with external abnormalities was found in this study.
Tissue distribution analysis of cerium in parental and pup tissues revealed that nano cerium dioxide was not detected in almost all of the samples. Only a few samples were slightly above the mean cerium content of blank samples, but this was also observed in the vehicle control and there was no correlation in cerium content among the tissues and dose groups.
The authors concluded that, under the experimental conditions of this study, no nano cerium dioxide-related adverse effects on the general systemic signs as well as on the reproductive performance or developmental toxicity was observed at doses up to 1000 mg/kg bw/day. Therefore, the NOAEL for systemic toxicity and reproductive performance of the parents can be established at 1000 mg/kd bw/day and the NOAEL for peri- and post-natal developmental effects in the pups can be set at 1000 mg/kd bw/day as well. In addition, cerium dioxide nanoparticles were not deposited in the parental or pup internal organs after repeated oral exposure.
In the prenatal developmental toxicity study, performed according to a method equivalent to OECD guideline 414, pregnant CD-1 mice have been exposed at gestational day 9.5 to 0 (controls), 5 and 20 mg/kg bw nano cerium oxide, dispersed in a water/0.05% BSA solution, through pulmonary route using a single pharyngeal aspiration. The clinical situation of the animals (weight, behaviour, any sign of disease) was monitored until sacrifice on gestational day 18.5. Selected maternal organs (liver, lungs, placenta) were excised for macroscopic evaluation and uteri from pregnant females were collected, embryos and the corresponding placentas released and analysed for gross anatomical abnormalities. Number of resorptions, if present, were also recorded and classified as early or late depending on the morphology. Embryos were weighted and the crown-rump length measured. After macroscopic analyses, placentas and embryos were processed for paraffin embedding following standard protocols for microscopic analyses.
The authors found no overt toxicity in terms of miscarriage or malformations. No significant differences in the analysed parameters were observed among the experimental groups. A trend towards a higher number of resorption and a slightly reduced number of delivered embryos was observed with the highest dose of cerium dioxide but the difference was not statistically significant. There were no effects of treatment on crown-rump lenght of the pups.
The authors concluded that their data suggest a lack of serious embryotoxicity after pulmonary exposure to cerium dioxide.
From the perspective of regulators and policy makers, the authors concluded that their data imply that unintended pulmonary exposure to cerium dioxide nanoparticles at doses which can be realistically expected in occupational and environmental settings should not pose peculiar risk to pregnant women.
The robust study summary for this study was built with the information available to the public from the NANoREG Deliverable 4.14 Prenatal toxicity study with cerium oxide and MWCNT carbon nanotubes.
In the two studies reported above, performed with nanoforms of cerium dioxide, both studies have shown a general absence of maternal toxicity and peri/post-natal developmental or teratogenic effect when the test material was administered either by oral route, at a level up to 1000 mg/kg bw/day in rats or by pulmonary route (pharyngeal aspiration) at the highest administrable dose of 20 mg kg/bw in mice. In addition, cerium dioxide nanoparticles were not deposited in the parental or pup internal organs after repeated oral exposure.
Further developmental toxicity studies are not deemed necessary nor scientifically justifiable for (nano or bulk) cerium dioxide. This conclusion was based on all available relevant toxicological and toxicokinetic information on nano and bulk cerium dioxide (summarised in the waiving statement as well as in the extended waiving justification document attached to IUCLID Section 13).
Zirconium dioxide
No key experimental information on this endpoint is available for zirconium dioxide nor for any other zirconium compound. The results of the OECD 422 study performed with the read across substance zirconium acetate however offers supporting information. Since the results of an OECD 422 study alone cannot be considered sufficient for waiving further testing on this endpoint, an assessment has been made based on all available (relevant) information on zirconium dioxide (an insoluble zirconium compound) and several other zirconium compounds (including both insoluble (zirconium basic carbonate) and 'water soluble' zirconium compounds (zirconium dichloride oxide, zirconium sulfate, zirconium acetate)).The full evaluation has been presented in the extended waiving justification document attached to IUCLID Section 13.
The following elements were considered to support the conclusion that further testing (prenatal developmental toxicity study) is not scientifically justified for zirconium dioxide:
- Zirconium, whether from an insoluble or ‘water soluble’ zirconium compound, is extremely poorly soluble at environmentally and physiologically relevant pH levels.
- There is sufficient evidence indicating that zirconium is barely absorbed neither from the gastrointestinal tract nor after exposure via inhalation or contact with the skin and thus, the probability to reach the reproductive organs and the unborn offspring is considered to be extremely low.
- Zirconium compounds have a very low potential for causing toxicity, both acutely and long-term, and regardless of the route of exposure. Not much difference exists among the zirconium compounds considered in this evaluation, although there is some evidence (based on acute LD50 values) for ‘water soluble’ compounds to be slightly more toxic (may be due to the effect of the counter ions (acidification)). Read across from ‘water soluble’ to insoluble zirconium compounds therefore guarantees that the extrapolation is on the safe side.
- The available repeated dose toxicity studies did not report any adverse effects on specific organs (including reproductive organs), biochemistry, hematology, etc.
- So far none of the zirconium compounds tested has been found to be a genetic toxicant in vitro.
- An OECD 422 study with zirconium acetate did not reveal any adverse effects on reproduction or development of rats up to the highest dose tested (NOAEL >= 1000 mg anhydrous zirconium acetate/kg bw/d). This study can be extrapolated to zirconium dioxide.
- Due to the extremely low absorption and toxicity, it will be scientifically unjustified to perform a prenatal developmental toxicity study, since this study reasonably requires testing up to doses that cause maternal toxicity. Based on the evaluation and results mentioned above, it is considered acceptable to assume that effects on reproduction or development are not to be expected (if at all) at exposure levels well below the unbound values from the repeated dose toxicity studies.
- Finally, considerations on exposure were added to the argumentation (only occupational exposure, inhalation exposure never up to doses as high as the unbound NOAEC levels from the repeated dose toxicity studies – which are above the occupational exposure limits anyway, risk management measures advised in the guidance on safe use (local exhaust ventilation, respiratory protection), etc.).
Reaction mass of cerium dioxide and zirconium dioxide
Based on an assessment taking into account all available data as well as toxicokinetic considerations, it is considered scientifically not necessary nor justifiable to perform further developmental toxicity studies with the reaction mass of cerium dioxide and zirconium dioxide or either of its constituents. The extended waiving justification document is attached to IUCLID Section 13.
Justification for classification or non-classification
No classification for reproductive or developmental toxicity is warranted for the reaction mass of cerium dioxide and zirconium dioxide according to the CLP criteria based on the absence of relevant effects in studies performed on its constituents.
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