Hydroxido potassium zirconium carbonate, where there are 0, 1 or 2 hydroxide groups and 2, 3 or 4 potassium atoms and 2, 3 or 4 carbonate groups.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

27 October 2009 to 12 May 2010.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A GLP study performed to a standardised guideline with a sufficient level of detail to assess the quality of the submitted data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium Zirconium Carbonate

IUPAC Name:

Ammonium Zirconium Carbonate

Test material form:

not specified

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:HsdRccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for twelve days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 308 to 356g, the females weighed 189 to 222g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. A certificate of analysis was provided in the original study. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2ºC and 55 ± 15% respectively.

The animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

IN-LIFE DATES: From: 03 November 2009 To: 18 December 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

Preparation of Test Material:
For the purpose of this study the test material was prepared at the appropriate concentration as a solution in Distilled water.

Dosing procedure:
The test material was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 10 ml/kg/day of Distilled water. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Details on mating procedure:

Following two weeks of treatment, animals were approximately fourteen weeks of age and were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 1364-0013).

Due to the complex nature of the test material and its limited solubility, a specific quantitative method of analysis was not developed. The concentration of the test material formulations was determined using a gravimetric technique. Homogeneity was assessed visually and stability was sampled after storage at +4ºC in the dark for twenty seven days. These analytical methods were considered to be sufficient for the purpose of this study.
Results showed the formulation to be stable for at least twenty-seven days. The formulations were prepared weekly during the treatment period and stored at approximately 4ºC in the dark.

A sample of each test material formulation was taken and analysed for concentration of Ammonium Zirconium Carbonate at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The results indicate that the prepared formulations were within ± 5% of nominal values.

Duration of treatment / exposure:

Forty-five consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Daily, 7 days a week

Details on study schedule:

Chronological Sequence of Study:

Dose Groups 1 to 4

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iii) One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each dose group and analysed for haematological and blood chemical assessment.

iv) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

v) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

vi) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vii) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

viii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

ix) Additional blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.

x) Additional blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
± 5%

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Clinical Observations

All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

Functional Observations

Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural Assessments

Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait; Tremors; Twitches; Convulsions; Bizarre/Abnormal/Stereotypic behaviour; Salivation; Pilo-erection; Transfer arousal; Exophthalmia; Lachrymation; Palpebral closure; Hyper/Hypothermia; Skin colour; Respiration; Urination; Defecation; Tail elevation and Transfer arousal.

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests contained in the original report.

Functional Performance Tests

Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The evaluation period was thirty minutes for each animal. The time in seconds each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the
force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:

Grasp response; Touch escape; Vocalisation; Pupil reflex; Toe pinch; Blink reflex; Tail pinch; Startle reflex and Finger approach.

Bodyweights

Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Bodyweights were also recorded prior to termination.

Food Consumption

During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded between Days 1 and 5 post partum. Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during the premating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption

Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Pregnancy and Parturition

Each pregnant female was observed at approximately 08.30, 12.30 and 16.30 hours and around the period of expected parturition. Observations were carried out at approximately 08.30 and 12.30 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Haematology

The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed.

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry

The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea; Calcium (Ca++); Glucose; Inorganic phosphorus (P); Total protein (Tot.Prot.); Albumin; Albumin/Globulin (A/G) ratio (by calculation); Aspartate aminotransferase (ASAT); Alanine aminotransferase (ALAT); Alkaline phosphatase (AP); Sodium (Na+); Creatinine (Creat); Potassium (K+); Total cholesterol (Chol); Chloride (Cl-); Total bilirubin (Bili); Bile acids (Bile).

Oestrous cyclicity (parental animals):

Not performed.

Sperm parameters (parental animals):

Not performed.

Litter observations:

Litter Data

On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum. For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development

All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

Pathology

Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed after Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights

The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Liver, Thyroid, Brain, Ovaries, Uterus, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus.

Histopathology

Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin except where stated:
Adrenals, Oesophagus, Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides♦, Skin (hind limb), Eyes*, Spinal cord (cervical, mid-thoracic and lumbar), Heart, Spleen, Ileum (including Peyer’s patches), Stomach, Jejunum, Testes♦, Kidneys, Thymus, Liver, Thyroid/parathyroid, Lungs (with bronchi)#, Trachea, Lymph nodes (cervical and mesenteric), Urinary bladder, Mammary gland, Uterus & Cervix, Muscle (skeletal), Vagina.

♦ = preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately
48 hours later
* = eyes fixed in Davidson’s fluid
# = Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

All tissues were despatched to the testing laboratory for processing. The tissues from five selected control and 1000 mg/kg/day dose group animals, any animals dying during the study, and any which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown below from the remaining control and 1000 mg/kg/day were also processed: Ovaries, pituitary, prostate, seminal vesicles, testes, uterus & cervix, vagina, coagulating gland, epididymides and mammary glands.

In addition, sections of testes and epididymides from all Control and 1000 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE 84 Pathology computerisation system for tabulation and report production.

Postmortem examinations (offspring):

Pathology

All offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

Statistics:

Statistical Analysis

-Data were processed to give group mean values and standard deviations where appropriate.

-The following parameters were subjected to analysis:

Quantitative functional performance
Bodyweight
Food consumption during gestation and lactation
Pre-coital intervals and gestation lengths
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight
Offspring surface righting
Haematology, blood chemistry, adult absolute and bodyweight relative organ weights

See the field "any other information on materials and methods incl. tables" for a detailed description of the statistical procedures adopted.

Reproductive indices:

MATING PERFORMANEC AND FERTILITY
The following parameters were calculated from the individual data during the mating period of the parental generation.
-Pre-coital interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
-Fertility Indices, for each group the following were calculated:
Mating Index (%) = [No. of animal paired/No. of animals mated] x 100
Pregnancy Index (%) = [No. of animals mated/ No. of pregnant females] x 100

GESTATION AND PARTURATION DATA
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
-Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
-Parturition Index, the following was calculated for each group:
Parturition Index (%) = [No. of pregnant females/No. of females delivering live offspring] x 100

Offspring viability indices:

LITTER RESPONSES
The standard unit of assessment was considered to be the litter; therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

-Implantation losses (%), group mean percentile pre-implantation and post implantation loss were calculated for each female/litter as follows:
% pre-implantation loss = [(No. of Corpora Lutea – No. of implantation sites)/No. of corpora lutea] x 100
% post-implantation loss = [(Group no. of implantation sites – No. of offspring born)/No. of implantation sites] x 100

-Live birth and viability indices, the following indices were calculated for each litter as follows:
Live Birth Index (%) = [No. of offspring born/No. of offspring alive on day 1] x 100
Viability Index 1 (%) = [No. of offspring alive on Day 1/No. of offspring alive on Day 4] x 100

-Sex ratio (% males), was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Sex ratio = [No. of male offspring/Total no. of offspring] x 100

Results and discussion

Results: P0 (first parental generation)

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

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

ADULT OBSERVATIONS AND EXAMINATIONS

Mortality:
There were no unscheduled deaths.

Clinical Observations:
No clinically observable signs of toxicity were detected. Isolated episodes of increased salivation were detected in three males treated with
1000 mg/kg/day on Day 36, and red/brown staining of the mouth was observed in two males treated with 1000 mg/kg/day on Day 35. Such findings are often observed following the oral administration of an unpleasant tasting test material formulation and are considered not to be indicative of systemic toxicity. Red/brown staining around the eyes was evident in one male treated with 100 mg/kg/day on Day 24 and generalised fur loss was observed for one female treated with 1000 mg/kg/day. This observation was also observed for one female treated with 100 mg/kg/day and also for one control female. These findings are occasionally observed in laboratory maintained animals and are unrelated to treatment. No clinical signs were detected in animals of either sex treated with 300 mg/kg/day.

Behavioural Assessments:
Weekly open field arena observations did not reveal any treatment-related effects. All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests:
There were no toxicologically significance effects detected in the functional performance test results from treated animals when compared to controls. Males treated with 1000 and 300 mg/kg/day showed a statistically significant reduction in forelimb grip strength when compared to controls (P<0.01 and P<0.05 respectively). This was only seen in one of the three tests performed and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity; these findings are considered to be of no toxicological significance. No such effect was detected in females treated with 1000 or 300 mg/kg/day or for animals of either sex treated with 100 mg/kg/day.

Sensory Reactivity Assessments:
There were no treatment-related changes in sensory reactivity scores for treated animals when compared to controls. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological
importance.

Bodyweight:
No adverse effects on bodyweight gains were detected for treated males, in comparison to controls throughout the treatment period, or for treated females during the pre-mating or gestation phases of the study. A statistically significant reduction in bodyweight gains was evident during lactation for the 1000 mg/kg/day females, in comparison to controls (P<0.01). In the absence of any supporting effects on dietary intake or histopathological correlates this finding was considered to be of no toxicological significance.

Reproductive Performance:
Mating: There were no intergroup differences in mating performance for treated animals when compared to controls. All animals mated within four days of pairing. Statistical analysis of the pre-coital interval data did not reveal any significant intergroup differences.
Fertility: No treatment-related effects were detected in fertility for treated animals compared to controls. Pregnancy was achieved for all ten females treated with 300 and 1000 mg/kg/day and for nine control and 100 mg/kg/day females.
Gestation Length: No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All pregnant females showed gestation lengths between 22 to 23½ days. Statistical analysis of the gestation length data did not reveal any significant intergroup differences.
Litter Response: In total ten females from the 1000 mg/kg/day dose group, nine control and 100 mg/kg/day females and eight 300 mg/kg/day females gave birth to a live litter and successfully reared young to Day 5 of age. One female treated with 100 mg/kg/day and one control female failed to achieve pregnancy. One female treated with 300 mg/kg/day showed corpora lutea and an implantation site but failed to produce a litter and a total litter loss was evident for another female treated with 300 mg/kg/day. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

POSTMORTEM EXAMINATIONS

Organ Weights:
No treatment-related effects were detected for organ weight measurements from treated animals in comparison to controls. A statistically significant reduction in liver weight both absolute and relative to terminal bodyweight was evident in males treated with 300 mg/kg/day when compared to controls (P<0.05). In the absence of a dose-related response these minimal reductions were considered to be incidental and unrelated to treatment.

Necropsy:
No treatment-related macroscopic abnormalities were detected for treated adults in comparison to controls. One 1000 mg/kg/day male showed small epididymides, prostate, seminal vesicles and testes. One female from this treatment group had pale adrenals. A mass on the left horn of the uterus which appeared to be a placenta was seen in one 300 mg/kg/day female. One animal of either sex treated with 100 mg/kg/day and one control female had reddened lungs. Small seminal vesicles were seen in a further male from this treatment group. No macroscopic abnormalities were detected in the remaining animals. The effects observed in the adults are considered to be incidental findings of no toxicological significance.

Histopathology:
No treatment-related changes were observed. All histopathological changes seen among control and high dose animals were considered to be spontaneous in origin and unrelated to treatment. The following conditions warrant specific mention:

ADRENAL GLANDS: Cortical vacuolation was seen in a few controls and treated males and was of no toxicological significance in this investigation.

BONE MARROW: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.

KIDNEYS: Globular accumulations of eosinophilic material, as a consequence of excessive accumulation of alfa2-microglobulin in renal proximal tubular epithelial cells, are occasionally encountered as a spontaneous change in male rats.

LIVER: Scattered mononuclear cell foci were observed in a few control and treated animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered. Similarly, generalised hepatocyte hypertrophy was seen in a few animals as a spontaneous condition.

LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for all control and high dose animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are similarly not suggestive of significant respiratory disease. Foreign body granulomatous reaction was seen in the lung of two high dose females; this is likely to be a consequence of accidental instillation of the test material into the airways at dosing and is not a systemic effect.

SKELETAL MUSCLE: Mononuclear cell foci are commonly observed in the skeletal muscle of laboratory maintained rats and are of no toxicological significance at the incidences seen in this investigation.

SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and although there was a slightly greater incidence of higher grades of the condition among high dose female rats this was considered to be fortuitous and unrelated to treatment.

STOMACH: Mucosal atrophy and mucous cell hypertrophy/hyperplasia were seen for one high dose male but this was considered to be unrelated to treatment.

REPRODUCTIVE TRACT AND RELATED ORGANS

PITUITARY: No treatment-related changes were seen. Vacuolation of pars anterior cells is a common spontaneous finding in male rats.

TESTIS/EPIDIDYMIS: No treatment-related changes were seen. Spermatocoel granuloma formation is observed occasionally as a spontaneous condition in the epididymis of male rats.

SEMINAL VESICLES/COAGULATING GLAND: No treatment-related changes were seen. Reduced secretary content was seen for one high dose male.

PROSTATE: No treatment-related changes were seen. Reduced secretary content was seen for one high dose male and one 100 mg/kg/day male.

MAMMARY GLAND: No treatment-related changes were seen. Glandular hyperplasia was observed in the mammary tissue of the majority of females examined and is consistent with pregnancy and lactation.

OVARY: No treatment-related changes were seen. A cystic corpus luteum was seen for one high dose female.

UTERUS/VAGINA: Areas of foam cells, haemorrhage and pigment were seen in the myometrium and adjacent connective tissue of the uterus in the majority of females examined from control and high dose groups. These conditions are consistent with normal post partum uterine changes in the rat. Dilatation of the uterine horns is a normal cyclical change in female rats.

All other morphological changes in the above and remaining tissues were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

LABORATORY INVESTIGATIONS

Haematology:
No treatment-related effects were detected in the haematological parameters measured. On Day 14, males treated with 1000 mg/kg/day showed slight but statistically significant increases (P<0.05) in total leucocyte, eosinophil and neutrophil counts in comparison to controls. In the absence of any supporting changes identified on Day 42 of treatment or any histopathological changes to suggest an effect of treatment, these findings are considered to be of no toxicological significance. No such effect was evident in the 1000 mg/kg/day females, or animals of either sex treated with 300 or 100 mg/kg/day. On Day 4 post partum, females treated with 1000 mg/kg/day showed a slight but statistically significant increase (P<0.05) in mean corpuscular haemoglobin concentration and platelet levels in comparison to controls. All individual values were within normal ranges and in the absence of any histopathological changes to suggest an effect of treatment, these findings are considered to be of no toxicological importance. No such effect was evident in males treated with 1000 mg/kg/day or animals of either sex treated with 300 or 100 mg/kg/day.
 
Blood Chemistry:
No treatment-related effects were detected in the blood chemical parameters investigated. Pre-mating blood chemical analysis revealed a slight but statistically significant reduction in albumin levels for males treated with 1000 mg/kg/day when compared to controls (P<0.05). All the individual values were within normal ranges for rats of the age and strain used, as such, this finding was considered to have arisen incidentally. A slight but statistically significant increase in alkaline phosphatase was seen in the 100 mg/kg/day females during Day 14 in comparison to controls (P<0.05). In the absence of a dose related response this minimal increase was also considered to have arisen incidentally. Following the Day 4 post partum assessments, a statistical significant increase in albumin and creatinine levels was evident in the 1000 mg/kg/day females when compared to controls (P<0.05), whilst a statistical significant increase (P<0.05) in alkaline phosphatase was seen in the 100 mg/kg/day females. On Day 42 males treated with 1000 mg/kg/day showed a statistically significant reduction in alanine aminotransferase levels in comparison to controls (P<0.05). All the individual values were within normal ranges for the rats of the age and strain used and in the absence of any histopathological correlates these finding are considered to have arisen incidentally and were unrelated to treatment.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The concentration was verified to be ± 5% of the nominal concentration.

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Litter Size and Viability:
No treatment-related effects were detected. A reduction in implantation sites and litter sizes at 1000 mg/kg/day were seen but no differences in pre or post implantation loss or live birth and viability indices were detected. No statistically significant changes were detected therefore, the slight
differences are considered to be unrelated to treatment. There were no treatment-related changes in sex ratio for treated animals in comparison
to controls. No significant differences were detected in litter sizes and viability for treated groups when compared to controls. At termination, ten litters were evident in the 1000 mg/kg/day dose group, nine litters were evident for the control and 100 mg/kg/day dose groups and eight litters were evident in the 300 mg/kg/day dose group.

Bodyweights (growth and development):
No treatment-related effects were detected. A slight but statistically significant increase (P<0.05) in Day 4 group mean offspring bodyweights was evident in the 300 and 1000 mg/kg/day males, in comparison to controls. There was no difference in litter weights detected at these dose levels. This
finding is considered to be incidental and unrelated to treatment. There were no differences in litter weights or mean offspring bodyweights detected in the 1000 or 300 mg/kg/day females or animals of either sex treated with 100 mg/kg/day. No clinically observable signs of toxicity were detected.
No treatment-related effects on surface righting was detected.

Necropsy:
No treatment-related macroscopic abnormalities were detected. One female treated with 300 mg/kg/day produced a litter with two pups which had
reddened lungs. Another female from this treatment group had one small pup. One female treated with 100 mg/kg/day produced a litter with one small pup. One control female produced a litter with a small pup; another female had a pup with a bruised head. A further female produced a litter which had a pup with a swollen left forelimb. The macroscopic observations were considered to be low incidence findings occasionally observed in reproductive studies of this type, and not related to test material toxicity. One interim death pup in a 1000 mg/kg/day litter, six interim death pups seen in one 300 mg/kg/day litter and two interim death pups seen in one 100 mg/kg/day litter all showed autolytic changes. The finding observed in the interim death offspring were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test material. There were no abnormalities detected in the remaining animals.

Organ weights and histopathological findings are discussed together with the adult findings in the field "details on results (parental animals)".

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No treatment-related effects in the reproductive performance and offspring development up to day 4 of lactation were detected at dose levels of up to 1000 mg/kg/day. Therefore, the “No Observed Effect Level” (NOEL) was considered to be 1000 mg/kg/day for reproductive and developmental toxicity.

Executive summary:

A GLP-compliant study has been conducted in accordance with OECD Guideline 422 to determine the toxicity to reproduction caused by repeat dosing of the test material.

Rats were administered Ammonium Zirconium Carbonate via oral gavage for a period up to forty-five consecutive days. The dose range selected was 100, 300 and 1000 mg/kg/day prepared in distilled water. Animals receiving dose levels of up to 1000 mg/kg/day and their offspring did not result in any toxicologically significant effects. Therefore, the “No Observable Effect Level” (NOEL) for animals of either sex was considered to be 1000 mg/kg/day for systemic toxicity.