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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2012-12-06 to 2013-02-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed under Good Laboratory Practices (GLP) and according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) without significant deviation. A maximal reliability score of Klimisch 2 (reliable with restrictions) could be assigned to this study because it is used for read across purposes in this dossier.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
- Name of test material (as cited in study report): zirconium acetate solution
- Substance type: unknown
- Physical state: liquid
- Analytical purity: > 99% zirconium acetate anhydrous
- Composition of test material, percentage of components: aqueous solution containing 40.7% zirconium acetate anhydrous
- Lot/batch No.: 12/201
- Expiration date of the lot/batch: 2013-07-01
- Stability under test conditions: 8 days at room temperature in the concentration range of 10 to 100 mg/mL
- Storage condition of test material: room temperature
- Other: colourless, clear solution

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

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 cages 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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
other: actual ingested (zirconium acetate anhydrous)
Remarks:
Doses / Concentrations:
53, 159, 530 mg/kg bw/day
Basis:
other: actual ingested (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

Examinations

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 sarificial 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 visualize 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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(toxicologically irrelevant)
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
- 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.









Effect levels (P0)

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.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING)
- 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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).