Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on supporting substance, but according to OECD guideline 426 and GLP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Reference Type:
publication
Title:
DOUBLE-BLIND, VEHICLE-CONTROLLED RANDOMIZED TWELVE-MONTH NEURODEVELOPMENTAL TOXICITY STUDY OF COMMON ALUMINUM SALTS IN THE RAT
Author:
J. POIRIER, H. SEMPLE, J. DAVIES, R. LAPOINTE, M. DZIWENKA, M. HILTZ, D. MUJIBI
Year:
2011
Bibliographic source:
Neuroscience, Vol. 193, 13 October 2011, Pages 338–362

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 426: Developmental Neurotoxicity Study (adopted on October 16, 2007)
Deviations:
yes
Remarks:
Usual termination data from 60-70 days of age of test animals to one year; Aluminium concentrations additionally measured in selected tissues
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium citrate
EC Number:
250-484-4
EC Name:
Aluminium citrate
Cas Number:
31142-56-0
Molecular formula:
C6H8O7.xAl
IUPAC Name:
aluminum citrate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Aluminium Citrate
- Supplier: Chemos GmbH (Regenstauf, Germany)
- Appearance: white powder
- Purity: 9.3% Aluminium by mass from January 23, 2008 to March 26, 2008, 9.8% from April 2, 2008 to November 4, 2008, 8.7% from November 13, 2008 to February 4, 2009. Purity stated here was as per the certificates of analysis from the supplier for each lot.
- Deviations from the stated analysis are discussed in Section 7.2.5 of the Final report, 2009.
- Batch or Lot Numbers: 136832, 128064, 143266
- Storage: Room Temperature
- Method of preparation: Calculated mass of dry aluminium citrate powder was added to ca. 75% of calculated volume of boiling deionized water and stirred. Mixture was covered and heated to 96°C-boiling temperature until the test substance crystals were in solution. Mixture was allowed to cool to room temperature, pH was adjusted to 6-7 and filtered. This filtered stock solution was used for preparation of the actual doses.

Reference compound:
- Name or Code: Sodium Citrate USP
- Supplier: Sigma-Aldrich
- Appearance: powder
- Purity: 100.2% and 99.9%
- Batch Number: 097K0017, 028K0067
- Storage: Room Temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
- Supplier: Charles River Canada Inc.
- Number of Animals in the Study: The study began with 180 pairs (i.e. adult male and female). This number was used to ensure 100 litters of progeny were delivered within the allotted timeframe (5 consecutive days).
- Age at Initiation of Treatment: 14-16 weeks at breeding
- Weight Range 3 days prior to pairing (grams): Females: 242.5-333.4 (target 160-360) grams; Males: 335.4-470.8 (target 245-585) grams
- Sex: male and female
- Housing: With the exception of sire/dam pairings during the breeding period and dams with litters, animals were housed individually.
- Caging: Prior to breeding, sires were housed singly in ventilated caging. Dams were housed singly in conventional shoebox caging prior to breeding and during the gestation period, then with their pups during the lactation period. During the breeding period, sire/dam pairings were housed in wire bottomed cages. During lactation, pups were housed with the dam in conventional shoebox caging. After weaning, pups were housed individually in ventilated caging until postnatal day 120, when they were transferred to shoebox caging due to their large size. Harlan TEK-Fresh diamond soft bedding replaced standard corn cob bedding during the gestation and lactation periods, and when hematuria or diarrhea was observed, or other issues as specified by the veterinarian. All animals received plastic enrichment tubes only for environmental enrichment.
- Temperature: 18-26°C
- Relative Humidity: 30-70%
- Air Changes: ≥ 10 per hour in the room.
- Light Cycle: ~12 hr. light (6:00-18:00h)
- Diet during growth and lactation: 5K75 irradiated rat chow until arrival of custom diet, then Purina AIN-93G diet. Irradiated from at least five days prior to breeding. This diet is formulated as a growth/lactation diet and was fed to all animals until postnatal day 95-99. Samples of the diet were tested for aluminum, iron, manganese, copper, and zinc content. Diet levels of aluminum were 6-9 ppm (6-9 µg/kg diet) over the study.
- Diet for adult animals: Purina AIN-93M – Irradiated. This diet is formulated as a maintenance diet and was fed to all animals from postnatal day 95-99 (introduced on May 20, 2008) until the end of the study. Aluminum levels were approximately 7-7-5 ng/mL.
- Water was available ad libitum
- Pretreatment procedures: Rats were observed daily and body weights taken on the first day of the pre-test period. Rats were granted an acclimatization period of 9 days. Rats were randomly allocated to treatment groups using the SAS PROC PLAN procedure to minimize difference in body weight between groups. The PROC PLAN procedure was also used to randomly select sire and dam pairings for breeding. The allocation of animals to rack and position was performed using a Youden square so that there was approximately equal representation of groups within each shelf of the rack. The dams were housed separately from the sires. At the time of breeding the sires and dams were moved to wire-bottom cages to facilitate detection of vaginal plugs. A Youden square was generated to dictate placement of pairs within the wire-bottom cages. Upon the detection of a vaginal plug, or at the end of the breeding period, the sires were removed. Each inseminated dam was returned to her original shoebox cage, and remained in that room and position until postnatal day 1 or euthanasia. Only animals in apparent good health and within the specified age range were selected for randomization to treatment groups. Treatment groups were identified with letters A-E. Test facility staff were blinded as to which letter (A-E) denoted which of the treatment groups (1-5). That information resided with the unblinded ToxTest representative and test facility test item staff. The unblinded ToxTest representative aligned letter groups A-E with numbered groups 1-5 based on a single random number set from 1 to 5 generated by the ‘research randomizer’ found at www.randomizer.org/form.htm. When blinding was broken, the study director acquired the original group alignment form from test facility QA. Blinding was broken once draft contributing scientist reports had been acquired from the principal investigator (metals analysis), the statistician and the pathologist.
- Identification of the Animals: Ear tags and microtatoo
- Identification Numbers: Dams and sires were ear tagged within 3 days of arrival at the facility. Pups were identified on postnatal day 4 with a microtattoo on the pads of the feet. Pups 1 and 5 were ear tagged the day before weaning, the rest on the day of weaning. If ear tags fell out during the course of the study, some of the animals received tattoos on their tails. Otherwise, identification was made by cage card.
- Identification of the Cages: Cage cards

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
- Volume administered: assumed 120 mL/kg/day fluid consumption
- On gestational day 6, dosing of rat dams began with formulated test or reference item, i.e. aluminum citrate, sodium citrate or nothing, added to the drinking water at prescribed concentrations. From that day forward, the test item was administered to groups of pregnant animals during gestation, lactation, and to offspring during post-weaning, through to post-natal day 364.
- The required mass of dry aluminium citrate was added to about 75% of the necessary volume of boiling deionised water on a hot plate (with stirrer). The mixture was then covered and heated to 96ºC until all the aluminium citrate was dissolved. After allowing the mixture to cool to room temperature, the pH was measured and adjusted to between 6 and 7 using sodium hydroxide and hydrochloric acid. The volume was then brought to a known value using deionised water to produce a “stock solution”. The stock solution was then filtered (0.45 µm) and stored in an interim vessel. Formulations were prepared weekly and stored in a plastic carboy at ambient temperature. To produce the dosing solutions, a calculated volume of the filtered stock solution was measured into a carboy and diluted by the required amount with deionised water. The pH of the final dosing solution was measured to ensure that it was in the required range of 6 to 7. Dosing solutions were transported to the animal test facility in 18L plastic carboys.
Details on mating procedure:
Sires and dams were allocated to breeding pairs by using the SAS PROC PLAN procedure. The rats were allowed to breed for up to five consecutive nights. Breeding took place in wire mesh cages, which allowed vaginal plugs to collect on a tray below the cage which allowed more reliable detection of insemination. During the breeding period, female animals were checked daily for the presence of vaginal plugs (indicating insemination). The date of breeding (i.e. insemination) was defined as the day when a vaginal plug was first detected. Upon detection of a plug, breeding pairs were separated.
Duration of treatment / exposure:
On gestational day 6, the test item was administered to groups of pregnant animals during gestation, lactation, and to offspring during post-weaning, through to post-natal day 364 for cohort 4 (see scheme below).

Dams: Treated from GD 6 to PND 21.
Pups (males and females): Treated from PND 22 to PND 364 in cohorts according to the following scheme:
Cohort treatment scheme:
Cohort 1 – GD 6-21, PND 1-22
Cohort 2 – GD 6-21, PND 1-64
Cohort 3 – GD 6-21, PND 1-120
Cohort 4 – GD 6-21, PND 1-364

Frequency of treatment:
drinking water available ad libitum (daily, 7 days per week.)
Duration of test:
1 year
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 100, 300 mg Al/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
Dams: 20/group;
Offspring: 10-20 females and 10-20 males/group;
Litters: 20 litter/dose.

Beyond the treatment group allocations, dams (and their litters) were grouped according to day of delivery. This grouping allowed combining data according to postnatal day, later used in the analysis. After the end of the delivery week, litters were randomly distributed across litter groups
Control animals:
yes, concurrent vehicle
other: A citrate control group received sodium citrate, the citrate content being equimolar to that in the group receiving 300 mg Al/kg bw/day
Details on study design:
DOSE SELECTION:
Doses were selected based on the results of a previous study which was undertaken to determine the maximum tolerated dose (MTD) of aluminium citrate administered in the drinking water for a period of 90 days in Sprague-Dawley rats, and the maximum solubility of aluminium citrate in water (high dose). The number of dose levels and dose spacing was according to guideline.
All pups continued to receive aluminum citrate, sodium citrate or nothing in the drinking water at the same dose level assigned to their respective dams.

The proportion of dams from each treatment group delivering on a given day could not be determined prospectively. Therefore, to improve the
chance of having a balanced distribution of deliveries, and hence of cages housing dams and their litters, extra breeding pairs (180 total versus 100 required) entered the study and after the end of the delivery week, litters eligible to enter the study were randomly picked to even the distribution of day over day litter groups as much as possible. Ideally the day over day distribution of deliveries, if even, would have yielded four litters per treatment group per day. In fact, fewer than expected dams delivered on the first parturition day and litters from subsequent days were allocated into the study to provide the total required number of litters from each treatment group. Beyond the need to compensate for undersubscribed groups, dams that delivered in excess of the daily quota were not included in the study.

EUTHANASIA OF ADULT ANIMALS:
Sires not allocated out of the study were euthanized CO2 asphyxiation after insemination or at the end of the breeding period. Dams that showed no evidence of insemination were euthanized by CO2 asphyxiation or reallocated out of the study at the end of the breeding period. Dams that showed insemination (i.e. a vaginal plug was detected) but did not deliver litters within the parturition period were euthanized by CO2 asphyxiation at the end of the parturition period. Dams and litters in excess of the number required for their group (i.e. all litters that were delivered over and above the 20
required in that group) were euthanized by CO2 asphyxiation (barbiturate overdose was used in error on 18 animals) or reallocated out of the study.

PUPS:
Litter Normalisation
At PND 4, litters were normalized to 4 males and 4 females. To ensure that the litter normalization progressed without bias, the following procedure was implemented if there were greater than 4 pups of a given sex present within a given litter and the litter size was of at least 8 pups prior to normalization. Any litters comprised of fewer than 4 pups per sex were removed from the study. Ice cube trays containing 14 numbered wells were used. Sets of random numbers (from 1 to 14) were generated by the ‘research randomizer’ (www.randomizer.org/form.htm). A total of 400 random number
sets was generated. The generation of 400 random number sets allowed for 2 sets of 180 to be allocated to each litter (1 for males, 1 for females) plus 40 extra sets (to be used in cases where litter normalization had to be re-executed due to a procedural error or unforeseen circumstances). Random number sets were printed and transferred to the animal rooms. After use, the random number sets were included in the study file. At the beginning of the litter normalization, dams were removed to a holding cage. Pups were separated by sex within each litter. Female pups were placed in the wells of an ice cube tray in order (i.e. well 1, well 2, well 3... well 14). When all female pups had been placed in wells, the technician began to select pups based on the random number sets. The technician read the random number sets from left to right. The first number caused the technician to observe the corresponding well in the ice cube tray. If no pup was present in that well, the number was crossed off the ‘research randomizer’ printout and the technician proceeded to the next number. If a pup was present in the observed well, that pup was considered part of the normalized litter and maintained in the ice cube tray. The number was then circled on the ‘research randomizer’ printout. This process continued until 4 female pups have been selected. All animals not selected for inclusion into the normalized litter were removed to cull cages separated into treatment groups. Extra pups (4 males and 4 females per treatment group) from each treatment group were randomly chosen for whole body aluminum, iron, manganese, copper and zinc assay. Euthanasia was by decapitation for these animals and also all other culled pups. Pups selected to be part of the normalized litters were maintained in the ice cube tray for tattooing. After identification, pups were returned to the “home cage” with the dam. This process was repeated for the male pups from the same litter. After each set of random numbers had been used, the number of the dam and the sex of the pups, for which the set was used, was written next to the set number. Four days after birth, beyond normalization to 4 males and 4 females per litter, pups were assigned by number to each of 4 cohorts sacrifice day groups associated with milestone observations and sacrifice. Litters were further allocated to subgroups for regular necropsy (A) and perfusion fixation (B). Because the litters were already randomized, every second litter was assigned to the perfusion fixation subgroup. Pups were weighed. Note that these allocations were in addition to the groupings by delivery day described above for the dams. Excess pups in a litter were euthanized by decapitation as described above, except that measurements of total body aluminum, copper, iron, manganese and zinc levels were made on tissues from 4 animals per sex per dose level at Postnatal Day 4 from excess pups eliminated during litter size adjustment. These carcasses were identified by treatment group, birth date and dam identification saved frozen at below -10° C, and sent to test site for analysis. Fluid consumption was monitored. Daily observations were made on all pups. Pups were weaned at postnatal day 22 by moving them to individual ventilated caging and another Youden square was used to determine distribution of the pups within the rack.

Allocation to Cohort
Also on PND 4, one pup per sex and normalised litter was assigned by number to each of 4 cohorts (Cohort 1- PND1- 22, Cohort 2 – PND 23-64, Cohort 3- PND 65- 120, and Cohort 4 – PND 121- 364) associated with observations, examinations and sacrifice.

In addition to treatment group allocations, dams (and their litters) were also grouped according to day of delivery to facilitate scheduling of the different procedures.

Blinding
Assessors were blinded to treatment group. Treatment groups were identified with letters - Group A (30 mg Al/kg bw/day, Low dose group), Group B (Na citrate group), Group C (Control group), Group D (100 mg Al/kg bw/day, Mid dose group), and Group E (300 mg Al/kg bw/day, High dose group).

STATISTICAL ANALYSIS:
Statistical analyses were conducted using SAS® Release 9.1 for Windows. Data collected on dams and pups was analyzed separately and all statistical analysis on the pups was performed separately for each sex. Analyses for pups were done separately for each test day group unless specified otherwise. Statistical significance was declared when P ≤ 0.05.

Dams:
Body weights on dams from gestation day 6 (pre-dose) were compared between groups using a 1-way analysis of variance. Repeated measures analysis of variance was used to compare dam body weights (g) and average daily fluid consumption (mL) over time between groups. The body weight and fluid consumption analyses were each a single analysis covering both the gestation and postnatal periods. For body weights, the dam body weights from gestation day 6 (the first day of dosing) were included as a covariate in the model. Actual dosage received (in mg/kg) by the dams was compared between groups over time using repeated measures analysis of variance techniques. This analysis was completed following un-blinding. Analysis of variance (or a categorical procedure depending on the distribution of the data) was used to compare groups in terms of gestational age at time of delivery. If there were significant differences between groups in terms of gestational age at time of delivery, then adjustments had to be made to other analyses to control for these differences. The Functional Observation Battery (FOB) scores for dams were analyzed differently for the gestation and postnatal periods. For rank and ordinal data, the Cochran – Mantel – Haenzsel row mean statistic or Fisher’s exact test was used. For continuous variables, a two way ANOVA was used to analyze differences between groups, study days and group by study day interactions. For censored continuous variables, survival analysis procedures were implemented using Proc LIFEREG in SAS for each study day.

Pups:
• Repeated measures analysis of variance was used to compare pup body weights (g) and average daily fluid consumption (ml) during the entire study period (for body weights, postnatal days 4, 8, 11, 15, 17, 22, 29, and biweekly thereafter, and for fluid consumption, postnatal days 22 and weekly thereafter). Pup body weights up to postnatal day 22 were compared between litters, treatment groups and sacrifice day groups using repeated measures analysis of variance. Pup body weights following weaning (after postnatal day 22) were also analyzed separately using a repeated measures analysis of variance with the body weight at postnatal day 22 included as a covariate in the model. This second analysis of pup body weights allowed for a better controlled comparison of body weights during the time period for which we are able to calculate the dosage each pup received. Up until postnatal day 22, the amount of dose each pup was receiving via lactation or direct fluid consumption was not known.
• In addition, a growth curve was fitted to each of the cohort 4 pup’s body weight data using the generalized logistic model. From this curve, asymptotic maximum body weight, days to 50% of maximum body weight, and maximum growth rate were calculated. These parameters were compared between groups using a generalized least squares procedure.
• Actual dosage received (in mg/kg) by the pups following weaning was compared between groups over time using repeated measures analysis of variance techniques. This analysis was completed following un-blinding.
• Developmental landmarks (i.e. day of vaginal opening for females, or day of preputial separation for males) were compared between groups and litters using an analysis of variance.
• The Functional Observational Battery (FOB) scores for cohort 4 pups were analyzed using the Cochran-Mantel-Haenszel’s row mean score or Fisher’s exact test controlling for study day as a stratification factor for descriptive and rank order data. For continuous data, repeated measures ANOVA including group, study day and the 2-way interaction as factors was used. For parameters with censored data points, survival analysis procedures (Fixed effects partial likelihood, FEPL) accounting for repeated measures were implemented instead of ANOVA to account for the censoring. Neonatal (from postnatal days 5 and 11), juvenile (from postnatal day 22) and adult pup FOB data (from postnatal day 36, 45, 56 and every two weeks thereafter) were analyzed separately. Two measurements were taken for forelimb and hindlimb grip strength, however only the maximum reading was analyzed.
• T-maze (for cohort 1): The first arm entered (i.e. blocked or unblocked) was compared between treatment groups using Chi-square tests. In addition, the frequency of alternation was summarized by treatment group.
• Morris Water Maze (for cohorts 2, 3 and 4): Latencies (i.e. the time it took to reach the platform) in the training session and the visible platform study were analyzed using a repeated measures analysis. The search category data from the visible platform study and the probe test were compared between treatment groups using Fisher’s Exact test.
• For Motor Activity assessment (from Day 23, 64, 120 and 364 groups), total ambulatory counts were analyzed using repeated measures analysis of variance with treatment group, interval and the 2-way interaction included as factors. The log transformed mean +/- SE ambulatory counts were plotted for each of the 12 intervals over time for each treatment group to assess whether asymptotic levels were reached indicating habituation. Note: Even though the data system records fine events, only the ambulatory activity (Xa total plus Ya total) events were used in data analysis. Fine events were recorded but not reported.
• For the Auditory Startle studies (from Day 23, 64, 120 and 364 groups), response amplitudes were compared between treatment groups (to evaluate treatment effect) and blocks (to look for evidence of habituation) using either parametric or non-parametric analysis of variance techniques depending upon the distribution of the data. The data were normalized via logtransformation.
• Hematology, clinical chemistry, and coagulation parameters (for cohorts 1, 2, 3 and 4) were analyzed using a 1-way analysis of variance to test for between treatment group differences. The exceptions to this approach were TBIL (Clinical chemistry) and NUC_RBC (Hematology), which were analyzed using Cochran – Mantel – Haenszel test. Some of the variables were log transformed to achieve a normal distribution and these varied depending on the sacrifice day group analyzed. Note: coagulation data was only collected from cohort 2, 3 and 4 animals.
• Brain weights (from cohorts 1, 2, 3, and 4) were analyzed as absolute weights and as a proportion of total body weight using a one-way analysis of variance to compare between treatment groups. Where the variables were not normally distributed, a non parametric ANOVA was used if single transformation did not achieve the intended normalization.
• Aluminum concentrations (from cohorts 1, 2, 3, and 4) in the brain (cortex, cerebellum and brainstem), spinal cord (cervical and thoracic regions), liver, bone (right femur) and blood were compared between treatment groups using one way ANOVA after log transformation of the non-normal variables.. Iron, manganese, copper and zinc concentrations in the brain and spinal cord were also compared between treatment groups using the same procedures. Correlation analysis (Pearson/Spearman) of the concentrations was done between tissues and between the different metals within a tissue.
• For all parameters which were analyzed using analysis of variance techniques, if the model revealed statistical significance (P≤0.05), Tukey-Kramer adjusted comparisons were used to determine if pair-wise differences existed.

Examinations

Maternal examinations:
All dams underwent daily morbidity and mortality checks while in the study, and underwent FOB examinations on gestational days 7 and 13, a clinical examination on the day of delivery, and FOB examinations on postnatal days 3 and 10. Body weights on all dams were taken on days 6, 13, and 20 of gestation and on postnatal days 8, 15, and 22. Fluid consumption was monitored in all dams on gestational days 6, 13, 20, and then on postnatal days 1, 8, 15, and 22. The period between gestational day 20, and postnatal day 1 varied from dam to dam as the gestational period varied from dam to dam. The variation in gestational length also incurred variability in the commencement of lactation. An additional layer of variability was added to the data as lactation is expected to raise fluid consumption dramatically. Given these sources of variability, fluid consumption data for the period between gestational day 20 and postnatal day 1 was not analyzed. The data, however, was still collected and included in the study file.

The FOB examination included the following parameters:

BRIEF HOME-CAGE OBSERVATIONS (PART 1):
- posture
- involuntary movements: tremors, convulsions, wasting, palpebral closure

HANDLING OBSERVATIONS (PART 2):
- ease of removal
- ease of handling
- salivation
- piloerection
- muscle tone
- eyes (red crusty deposit, conjunctivitis, ocular exucate, opacity, lacrimation, fur appearance, mouth and nose deposits)

OPEN FIELD OBSERVATIONS (PART 3):
- posture
- involuntary movements: tremors, convulsions
- abnormal motor movements: writhing, circling, bizarre behaviors, stereotypic behaviors, gait, total gait score, rearing, vocalizations, arousal level, urination, urination/characteristics, defecation, defecation/characteristics

SENSORY AND NEUROMUSCULAR OBSERVATIONS (PART 4):
- approach response
- startle response
- tail pinch
- pupil size/response
Ovaries and uterine content:
No data.
Fetal examinations:
Not applicable.
Statistics:
See section "Further details on study design".
Indices:
No data.
Historical control data:
No data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL OBSERVATIONS:
- Mild clinical signs observed in all treatment groups, including the control and Na citrate group. Mainly mild dermatological lesions such as alopecia and porphyrin staining in the dams or bruising in the pups. 8 of the high-dose dams developed diarrhea (likely treatment-related).
- 1 dam in the Na citrate group stopped nursing, and she and the pups were euthanized early. The loss of this animal may have bee normal attrition, although it did sustain lesions from fighting during the mating period and an occult infection cannot be ruled out.

BODY WEIGHTS:
There were no significant differences between mean body weights of controls and treated groups, with the exception of postnatal day 15, where the Na citrate group mean was 7.3% less than controls (p=0.021). The Na citrate group body weights were also significantly less than those of the low dose group overall, and on postnatal days 8 and 15.

GESTATION LENGTH:
No significant differences in gestational length among the treatment groups.

FLUID CONSUMPTION:
During gestation and lactation, low dose animals consumed significantly more fluid than the Na citrate group (p=0.011) and the water control group (p= 0.0028). The mid-dose group consumed significantly more than the Na citrate (p<0.0001), water control (p<0.0001) and high dose groups (p=0.023). These significant differences were observed during weeks 1 and 2 (gestation days 6-20).
Mean fluid consumption:
- Na citrate group: 26.2-29.3 mL/day or 66-76 mL/kg bw/day (during gestation); 35.1-68.0 mL/day or 106-213 mL/kg bw/day.
- low dose Al group: 35.9-43.7 mL/day or 103-108 mL/kg bw/day (during gestation); 40.1–60.9 mL/day or 114–177 mL/kg bw/day.
- mid dose Al group: 42.0–45.2 mL/day or 112 - 123 mL/kg bw/day (during gestation); 40.9–69.0 mL/day or 136-201 mL/kg bw/day.
- high dose Al group: 27.4–31.3 mL/day or 78 - 80 mL/kg bw/day (during gestation); 39.7–70.2 mL/day or 120-211 mL/kg bw/day.

As increased fluid intake was not observed in the high dose group, the effect was considered not treatment-related.

DOSAGE LEVELS:
To assess the dosage levels, body weights were averaged over the weekly period, and these average weights used in the calculation of dosage for the week.
Mean dosage levels:
- low dose group: 25.9-27.0 mg Al/kg bw (during gestation, 10 – 14% below target of 30 mg/kg); 28.6–44.5 mg/kg (during lactation, range of 5% below to 50% above target of 30 mg/kg).
- mid dose group: 94.3–102.0 mg Al/kg bw (during gestation, 6% below to 2% above target of 100 mg/kg); 99.9 – 165.2 mg/kg (during lactation, 100% of target to 65% above target).
- high dose group: 195.4–199.9 mg Al/kg bw (during gestation, 35% below to 33% below target of 300 mg/kg); 298.6–523.3 mg/kg (during lactation, 100% of target to 74% above target).

Althoug the actual dosage was not 100% of target through gestation and lactation, it followed fluid consumption which varied similarly in all groups (according to the physiological needs of the dams), and group differences in dosage were maintained.

FOB ADULT DAM:
Overall, there were few group differences among the parameters measured. The only statistically significant group differences were as follows:
- Hind limb grip strength values for the Na citrate group were significantly less (p=0.0047) than those of the low and high dose groups in the gestational observations.
- Forelimb grip strength values for the controls, low and mid dose group (but not the high dose group) were significantly lower than those of the Na citrate group, and values for the mid dose group were also lower than those of the high dose group (but neither were different from the low dose group value, which lay between the high and mid dose values) in the postnatal observations.

All in all, there was no consistent evidence for any effects of test item on FOB characteristics in the dams.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: general maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: neuromuscular effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

RESULTS FOR PUPS

Mortality:

Animals from each treatment group and cohort died or were euthanized prior to their planned sacrifice dates, as demonstrated in the following table:

 

    Females

    Males

 

 Died

 Euthanized

Died 

 Euthanized

Water control

 4

 Low dose Al group 

 Mid dose Al group

 High dose Al group

37 

 Na citrate group

The main cause for mortality was urinary tract pathology in the high dose group, especially the males, but also affecting some females. Urinary tract pathology included hydronephrosis, ureteral dilation, obstruction and/or presence of calculi (chalky white concretions and deposits varying from sand-like material through to large, irregular stones measuring up to 4 mm diameter). Obstruction by calculi occured at any level of the urinary tract, including the urethra, bladder, ureters, and renal pelvis. Dilation of the ureters and hydronephrosis were found in both partially and completely obstructed rats. Several rats died, presumably due to hyperkalemia, especially high dose group males, where urinary calculi blocked the urinary tract at various levels, but also a few animals from the mid dose group and a few random ones from other groups also had urinary tract lesions.

Rats with urinary tract lesions of hydronephrosis, ureteral dilation, obstruction and/or presence of calculi are indicated in the following table:

 

 

          Collection Time

 Treatment

 Sex

 Cohort 1

 Cohort 2

 Cohort 3

 Cohort 4

 Na citrate

 M

 0

 1

 0

 0

 

 F

 0

 0

 1

 0

 Water control

 M

 0

 0

 0

 0

 

 F

 0

 1

 0

 0

 Low dose Al group

 M

 0

 0

 0

 1

 

 F

 0

 0

 0

 0

 Mid dose Al group

 M

 0

 3

 1

 0

 

 F

 0

 1

 0

 0

 High dose Al group

 M

 0

 11

 7

 5

 

 F

 3

 2

 3

Some normal attrition was encountered throughout the study, with a number of common spontaneous pathologies appearing without pattern. In particular, no treatment-related nervous system pathologies were found.

Clinical observations:

Cohort 1:

- High dose Al group: abdominal distention observed in a few animals, a few animals were small and cold.

Cohort 2:

- Na citrate group: 3 animals thin with poor hair coat (1 was euthanized)

- water control group: 1 female bloated and had diarrhea (was later euthanized)

- high-dose Al group: 1 female and 7 males were thin, had diarrhea, mild dehydration and poor hair coat. Additional abdominal distention in one of the males.

Cohort 3:

- Na citrate group: 3 animals thin with poor hair coat.

- high dose Al group: 5 females and 9 males had diarrhea. Hematuria in 1 female. Urinary tract pathology in several males.

Cohort 4:

- mid dose Al group: 1 animal limping. Seizures observed in 2 females and 3 males.

- low dose Al group: 2 animals limping

- Na citrate group: 3 animals limping. Seizures observed in 1 female. 2 females and 5 males with hematuria.

- water control group: 2 animals limping. Seizures observed in 1 female.

- high dose Al group: 1 female with hematuria. Several males with signs of urinary tract pathology. Seizures observed in 2 females (one later diagnosed with pituitary adenoma).

The limping was considered to result from trauma from the foot splay procedure and not be treatment-related. The incidence of seizures was too low to conclude whether they might have been treatment-related. Diarrhea and bloat, thinness, poor hair coat and hematuria were considered to be primary from the treatment or secondary to renal failure due to urinary tract pathology. After about half of the high dose males either died or were euthanized due to the severity of clinical signs, the remaining high dose males were euthanized for humanitarian reasons.

Body weights:

  • Pre-weaning phase: Analyses using the data from all cohorts combined showed no significant differences between the cohorts in body weights in the pre-weaning phase. Litter was also included in the analyses. A significant effect of litter was observed in both male and female pups. Results of pair-wise comparisons between treatment groups in the female pups showed that Na-citrate and high dose groups had significantly lower pre-weaning body weights than the control and low-dose groups (low dose v Na-citrate, p=0.0007; low dose v high dose, p=0.0398; control v Na-citrate, p<0.0001; control v high dose, p=0.0072). In the male pups, the low dose group had significantly greater body weights than the Na-citrate group (p=0.0004) and the high dose group (p=0.0239). The control group mean body weights were significantly greater than the Na-citrate group (p<0.0001) and also significantly greater than the high dose group (p=0.0051). The mid-dose group mean body weight was significantly greater than the Na-citrate group (p=0.0405).
  • Post-weaning phase:
    Day 23 cohort: Na-citrate group animals were significantly lighter than the low dose (p=0.0348 for females, p=0.0014 for males) and the control group (p=0.0305 for females, p=0.0033 for males) animals.
    Day 64 cohort (females):High dose animals were significantly lighter than all the other dose groups. The group x Study Day interaction term was significant. On Study Days 43 and 56, the high dose group was significantly lighter than all the other groups.Day 64 cohort, males:The Na-citrate group was significantly lighter than the low dose and the control groups (p=0.0008, p<0.0001, respectively). The group x Study Day interaction term was significant. On Study Day 43, the high dose group was significantly lighter than all the other treatment groups (all p<0.0001). The Na-citrate group was also lighter than the control group (p=0.0184) on this day. On Study Day 56, the high dose group was significantly lighter than all the other treatment groups (all p<0.0001); the mid-dose group was also significantly lighter than the control group (p=0.0211). The Na-citrate group was significantly lighter than the low dose (p<0.0001) and mid-dose (p=0.0003) groups on this study day also. Day 120 cohort, females:The effect of group was significant (p<0.0001) and pair-wise comparisons showed that the high dose group was significantly lighter than all the other groups (p <0.0001, p=0.0002, p=0.0151, and p=0.0002 for comparisons with the control, low-dose, mid-dose and Na-citrate groups, respectively). Day 120 cohort, males:The effect of group was significant (p<0.0001) and pair-wise comparisons showed that the Na-citrate group and mid-dose groups were significantly lighter than the control group (p=0.0011 and p=0.0016, respectively). The Na-citrate group was also significantly lighter than the low dose group (p=0.0203). Pre-dose body weight was included as a covariate in the analyses. The Group x Study Day interaction term was significant. In pair-wise comparisons, the high dose group was significantly lighter than the other treatment groups on Study Day 43, 56, 70, and 84. The Na-citrate and mid-dose groups were significantly lighter than the control group on Study Days 70, 84 and 98. Day 364 cohort, females:The effect of group was significant (p=0.0008) and pair-wise comparisons showed that the high dose group was significantly lighter than the control and mid-dose groups (p=0.0015 and p=0.0032, respectively) but not the low dose group. The group x Study Day interaction term was significant. The high dose group was significantly lighter than the control group on Study Days 294, 308, 322, 336, 350 and 364. The Na-citrate group was significantly lighter than the control on Study Days 322, 336, 350 and 364.Day 364 cohort, males (note: males euthanized at Day 84):The effect of group was significant (p=0.001) but there were no significant pair-wise differences between the control, low-dose, mid-dose, and Na-citrate groups. The group x Study Day interaction term was significant. Pair-wise comparisons showed that the high dose group was significantly lighter than the control and low-dose groups (p=0.0027 and p=0.0016, respectively) on Study Day 70. On Study Day 84, the high dose group was significantly lighter than the control, low-dose and Na-citrate groups.

In conclusion, the results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. An effect of Na-citrate was observed in the female pups. The high dose of Al citrate and sodium citrate administrations are therefore considered to have had adverse effects on the body weights and growth parameters of female pups.

Fluid consumption:

Day 64 cohort, females:The high dose group showed a significantly higher fluid consumption than the control, low-dose, mid-dose and Na-citrate groups (p<0.0001, p<0.0001, p=0.0356, p<0.0001, respectively). The mid-dose group fluid consumption was significantly higher than the low dose and control groups (p=0.0002 and p<0.0001, respectively). The control group consumed significantly more fluid than the Na-citrate group (p=0.0003).

Day 64 cohort, males:The mid-dose group showed a significantly higher fluid consumption than the control, low-dose, high-dose and Na-citrate groups (p<0.0001, p<0.0001, p=0.0432, p=0.0053, respectively). The high-dose group consumed significantly more fluid than the low dose and control groups (p=0.0449 and p=0.0044, respectively). The control group consumed significantly less fluid than the Na-citrate group (p=0.0257), unlike in the females.

Day 120 cohort, females:The high dose group showed a significantly higher fluid consumption than the control, low-dose, mid-dose and Na-citrate groups (p<0.0001 for all). The mid-dose group fluid consumption was significantly higher than the control group (p=0.0009). The control group consumed significantly less fluid than the Na-citrate group (p=0.0023) unlike in the females in the Day 64 cohort.

Day 120 cohort, males (high dose group missing):The mid-dose group showed a significantly higher fluid consumption than the control, low-dose, and Na-citrate groups (p<0.0001, p<0.0001, p=0.0252, respectively). The control group consumed significantly less fluid than the Na-citrate group (p=0.008).

Day 364 cohort, females:The high dose group showed a significantly higher fluid consumption than the control, low-dose, mid-dose and Na-citrate groups (p<0.0001, p<0.0001, p=0.0002, and p<0.0001, respectively).

The control group consumed significantly less fluid than the Na-citrate group (p<0.0001) and also significantly less than the low and mid-dose groups (p=0.004 and p<0.0001). The low-dose group consumed significantly less than the mid-dose and Na-citrate groups (both p<0.0001). Comparisons between groups on the different study days (43, 50, 56, 70, 77, 84, 91, 105, 112, 133, 140, 161, 175, 182, 196, 210) showed a consistent pattern of increased fluid consumption in the high dose group compared with the control.

Day 364 cohort, males (high dose group missing):The mid-dose group showed a significantly higher fluid consumption than the control and low-dose groups (p<0.0001 for both). The control group consumed significantly less fluid than the Na-citrate group (p<0.0001).

Day 364 cohort, males (to Study Day 91; high dose group included):The mid-dose group showed a significantly higher fluid consumption than the control and low-dose groups (p=0.0008 and p=0.0009, respectively). The control group did not differ significantly from the Na-citrate group.

Fluid consumption varied significantly between study days. In mid-dose males (Day 364 cohort), the mean fluid consumption during the first post-weaning week was 16.0 mL/day (equivalent to 171 mL/kg bw/day; 33% greater than in the controls); on study day 70 it was 36.4 mL/day (equivalent to 93 mL/kg bw/day; 63% greater than in the controls) and decreased on a per body weight basis until the end of the study. In high-dose females (Day 364 cohort), the mean fluid consumption during the first post-weaning week was 16.3 mL/day (equivalent to 207 mL/kg bw/day; 60% greater than the controls); on study day 112 it was 37.6 mL/day (equivalent to 130 mL/kg bw/day; 124% greater than the controls) and decreased on a per body weight basis until the end of the study. Overall, dosing of animals with aluminium citrate led to an increase in fluid consumption compared with the control animals. Dosing with Na-citrate was associated with a significant increase in fluid consumption relative to the controls in most cohorts, with the exception of the Day 64 cohort females (fluid consumption was significantly lower in the Na-citrate group) and the Day 364 males (no significant difference between the two groups). The animals’ fluid consumption varied with time and, in mature animals, was less than expected (120 mL/kg bw/day) with implications for the actual dosage of test item received.

Dosage levels:

The target dose for the low dose group was 30 mg Al/kg bw/day, for the mid-dose 100 mg Al/kg bw/day and for the high dose 300 mg Al/kg bw/day. The table below provides the arithmetic mean actual dose as a % of the target dose for 5 selected post-weaning weeks in the Day 364 cohorts.

Males

Group

Week 1

Week 7

Week 14

Week 28

Week 49

Low-Dose

134%

57%

37%

20%

17%

Mid-Dose

174%

84%

51%

28%

23%

High-Dose

165%

117%

-

-

-

Females

Low-Dose

145%

60%

57%

34%

33%

Mid-Dose

199%

74%

64%

38%

41%

High-Dose

205%

118%

93%

58%

42%

Despite the deviations from the target dose, the low-, mid- and high-dose groups showed the required trend of lowest to highest maintaining statistically significant group differences in dosage. For the majority of the study period, the actual dose received was less than the target dose in all treatment groups. Dosages in the Al-treated animals in cohort 4 were well below target as a direct result of the fact that the animals drank much less than the anticipated 120 mL/kg bw/day, especially as they fully matured.

Organ Weight:

The only organ weighed in this study was the brain:

- Day 23 cohort:Absolute brain weights did not differ significantly across treatment groups in males or females.

- Day 64 cohort:Absolute brain weights differed across the treatment groups in males (p=0.0003). The high dose group brain weights were significantly lighter than the controls (0.0007), low-dose (p=0.0256), and mid-dose (p=0.0003) groups. In females, the group effect was no significant (p=0.0868).

- Day 120 cohort:Group effects were significant in both males and females in the Day 120 cohort. In males, all adjusted p-values form the pair-wise comparisons were >0.05. In females, the difference between the high dose and the controls reached statistical significance (high dose brain weights less than in the controls, p=0.0346).

- Day 364 cohort:Absolute brain weights did not show significant effects of treatment group.

As the differences in brain weight were relatively small compared to differences in body weight, relative brain weights in this study tended to follow body weight. Overall, treatment did not appear to affect absolute brain weight.

Pathomorphology and Histology:

Necropsy Results

Urinary tract pathology (hydronephrosis, ureteral dilation, obstruction and/or presence of calculi) was an unexpected finding more prevalent in males and in the high dose group. The calculi (“chalky white concretions and deposits”) varied from sand-like material to stones up to 4 mm in diameter. Hyperkalemia was proposed by the pathologist as the cause of death of the animals with urinary obstruction. The chemical composition of the calculi was not determined.

The numbers of rats per cohort and treatment group that exhibited urinary tract pathology (hydronephrosis, ureteral dilation, obstruction and/or presence of calculi) are provided in the tables below:

Females

Day 23

Day 64

Day 120

Day 364

Control

0

1

0

0

Low-Dose

0

0

0

0

Mid-Dose

0

1

0

0

High-Dose

0

3

2

3

Na-citrate

0

0

1

0

Males

Day 23

Day 64

Day 120

Day 364

Control

0

0

0

0

Low-Dose

0

0

0

1

Mid-Dose

0

3

1

0

High-Dose

0

11

7

5

Na-citrate

0

1

0

0

 

Urinary tract pathology was a treatment-related effect. The only other treatment-related effect reported was watery, tan-coloured fluid in the digestive tract in some high dose animals, more frequently in the Day 64 group.

Histopathological examination of CNS tissue and muscle (microscopic)

Day 23 cohort:One female rat in the low dose group exhibited a necrotic neuron and a neuron with satellitosis in the basal ganglia. All other examinations were normal in all treatment groups.

Day 64 cohort:Control group – one male rat showed very mild inflammation of connective tissue around the sciatic nerve. Low dose group - All tissues were normal. Mid-dose group - All tissues were normal. High dose group - All tissues were normal. Na-citrate group - All tissues were normal.

Day 120 cohort:Control group – All tissues normal. Low-dose group - All tissues were normal. Mid-dose group - All tissues were normal. High-dose group - All tissues were normal.Na-citrate group - All tissues were normal.

Day 364 cohort:Control group – 3 females and 2 males had low numbers of neurons in the thoracic dorsal root ganglion, the neurons had small vacuoles. Low dose group - 1 female had a focal area of gliosis at one edge of the hippocampus; 4 female and 2 male rats had small numbers of neurons in the sections of thoracic dorsal root ganglion with small vacuoles in the cytoplasm. Mid-dose group – 3 females and 1 male had low numbers of neurons in thoracic dorsal root ganglion section and the neurons had vacuoles; a male had astrocytoma in the posterior hippocampus and 1 male had gliosis in one side of the central canal. High dose group - 3 female rats had low numbers of vacuolated neurons in the thoracic dorsal root ganglion; a vacuolated neuron was also observed in a lumbar spinal cord section from one rat, and from a section of cervical ganglion in another rat. Na-citrate group – 3 females and 2 males had low numbers of neurons in the thoracic dorsal root ganglion section and the neurons had vacuoles; 1 male rat had occasional spheroids in the white matter of the lumbar spinal cord.

Number of animals with vacuolated neurons in thoracic ganglia (Day 364 cohort):

Group

Sex

Day 364

Control

M

2

 

F

3

Low-Dose

M

2

 

F

4

Mid-Dose

M

1

 

F

3

High-Dose

M

n/a

 

F

3

The pathologist concluded that none of the lesions seen in the Day 364 group were treatment-related and, as they were also seen in the control group, were likely due to ageing.

Developmental landmarks:

Females

A significant (p<0.0001) group effect was observed. High dose female pups required significantly longer for vaginal opening to occur than the controls (p<0.0001), the low-dose group (p<0.0001), the mid-dose group (p<0.0001) and the Na-citrate group (p<0.0001). The Na-citrate group required significantly longer than the controls, low-dose and mid-dose groups for vaginal opening to occur (p<0.0001 for all). Litter was included in the model and contributed significantly to the variance. The mean number of days to reach vaginal opening was 31.3 (±2.1, sd) in the control group and 39.7 (±5.6, sd) in the high dose group.

Males

A significant (p<0.0001) group effect was observed. High dose male pups required significantly longer for preputial separation to occur than the controls (p<0.0001), the low-dose group (p<0.0001), the mid-dose group (p<0.0001) and the Na-citrate group (p=0.0205). The Na-citrate group required significantly longer than the controls, low-dose and mid-dose groups for preputial separation to occur (p=0.0034, p=0.001, and p=0.0017, respectively). Litter was included in the model and contributed significantly to the variance. The mean number of days to reach preputial separation was 39.6 (±2.1, sd) in the control group and 42.5 (±3.2, sd) in the high dose group. The number of days to to reach defined developmental landmarks (vaginal opening for females, preputial separation for males) are summarized in the following table:

Group

Sex

Day 364

Control

M

2

 

F

3

Low-Dose

M

2

 

F

4

Mid-Dose

M

1

 

F

3

High-Dose

M

n/a

 

F

3

In summary, delayed development of both male and female pups was observed in the high dose Al-citrate and Na-citrate groups. The effect is considered treatment-related. Whether the effect is secondary to decreases in body weight is not clear.

 

FOB:

Neonatal pup FOB:

Females:

Convulsions, salivation, and tremor were all zero in females. No significant group effects were observed for activity, foot-splay, lacrimation, posture, unusual appearance or unusual behaviour.

Males:

Convulsions, posture, salivation, tremor and unusual behaviour were all zero in males. Activity, foot-splay, lacrimation and unusual appearance did not exhibit significant differences across groups. The group effect approached statistical significance for foot-splay (p=0.0525) on PND11, with 4 of 20 in the high dose group receiving a rating of 1. The number of animals in the other treatment groups that received a rating of 1 versus 0 were 1 out of 20 for the controls, 0 out of 20 for the low dose group, 0 out of 20 for the mid-dose group and 1 out of 19 for the Na-citrate group.

Juvenile pup FOB:

Day 364 cohort

Females:

Righting reflex, muscle tone, and posture were all normal for the female pups. Lacrimation, salivation, unusual appearance, and unusual behaviour were all zero. Significant group effects were not observed for the other FOB parameters with the exception of forelimb grabbing (p=0.0278). The significant group effect was due to Na-citrate dosed animals holding on for significantly longer than low, mid and high dose Al-citrate animals.

Males:

Handling reactivity, lacrimation, salivation, muscle tone, posture, tremors, unusual behaviour, unusual appearance and righting reflex were all normal or zero for males. Significant effects were not observed for the other FOB parameters with the exception of No. of rears (p=0.0223). The significant group effect was due to Na-citrate animals exhibiting significantly fewer rears than the low dose Al-citrate group and the controls.

Adult pup FOB:

Day 364 cohort

Females

Significant group differences were observed for:

Wasting:High dose group had sig. more wasting than low dose group (p=0.0308), mid-dose group (p=0.0213) and controls (p=0.0042). Na-citrate group had sig. more wasting than low dose group (p=0.0345), mid-dose group (p=0.0233) and controls (p=0.0044).

Fur appearance:High dose group had sig. more abnormal fur appearance than controls (p=0.0001) and mid-dose group (p=0.0071) but the low dose group had sig. more abnormal fur appearance than the mid-dose group and the controls.

Mouth and nose deposits: High dose group had sig. more than controls and mid-dose group, but low dose and controls had sig. more than mid-dose group also.

Eye opacity:The low dose had sig. more than the other groups.

Salivation:Low and mid-dose had sig. more salivation than the high dose group and the controls.

Arousal (open field):The high dose group exhibited more arousal than the low dose group, the controls, and the Na-citrate group. The low-dose and mid-dose groups showed sig. more arousal than the controls.

Defecation:The high and mid-dose groups have more faecal boluses than the low-dose group, the controls and also the Na-citrate group.

Pupil response:The high dose group lacked response compared to the control and mid-dose groups. The low-dose and mid-dose groups lacked response compared with the control. The Na-citrate group also lacked response compared to the control.

Pupil size:The Na-citrate group is sig. more abnormal than the high dose group, the controls and the mid-dose group.

Rearing:All of the treatment groups exhibited significantly more rears compared with the controls. The low-dose group exhibited sig. more rears than the high dose group.

Tail pinch:The mid-dose group had sig. more abnormal reaction than the low dose, mid-dose, high dose and Na-citrate groups. The low dose group had sig. more abnormal reaction than the control group.

Urination:The Al-treated groups and the controls had sig. more urine pools than the Na-citrate group.

Urine characteristics:The low-dose, mid-dose and controls had sig. more urine pools and abnormal colour than the Na-citrate group.

Foot-splay:The low-dose group had sig. greater foot-splay measurements than the high dose group, the mid-dose group and the Na-citrate group. The control group had significantly greater foot-splay than the mid-dose group, the high-dose group and the Na-citrate group.

Forelimb grip strength:The controls had sig. greater forelimb grip strength than the mid- dose group (p<0.0001), the high-dose group (p=0.0066) and the Na-citrate group (p=0.0101). The low-dose group had sig. greater forelimb grip strength than the mid-dose group (p=0.0085).

Hind-limb grip strength:The controls had sig. greater forelimb grip strength than the mid- dose group (p=0.0007), the high-dose group (p<0.0001) and the Na-citrate group (p<0.0001). The low-dose group had sig. greater forelimb grip strength than the mid- dose group (p=0.0093), the high-dose group (p<0.0001) and the Na-citrate group (p=0.0012).

Males

Significant group differences were observed for:

Fur appearance:High-dose group had sig. more abnormal appearance than controls (p=0.0169), low-dose group (p=0.0016), and mid-dose group (p=0.0185).

Mouth and nose deposits:High-dose group had sig. more deposits than the low-dose group and the mid-dose group.

Eye opacity:Low-dose group had sig. more loss than controls, the mid-dose group and the Na-citrate group.

Red Crusty deposits:The mid-dose group had sig. more deposition than the controls and the Na-citrate group.

Exophthalmus:High dose group had sig. more eye bulging than the controls, the mid-dose group, and the Na-citrate group.

Piloerection:The mid-dose group had sig. more piloerection than the controls, the low dose group and the Na-citrate group.

Defecation:The Al-treated groups and the controls had more faecal boluses than the Na-citrate group. The low-dose group had fewer boluses than the controls, mid-dose group, and the high dose group.

Rearing:The high dose group exhibited sig. fewer rears than the Na-citrate group. The mid-dose group exhibited sig. more rears than the control and the low-dose groups. The low-dose group exhibited sig. more rears than the control group.

Tail pinch:The control group and the mid-dose groups had significantly more abnormal responses than the high dose group. The Na-citrate group had significantly more abnormal responses than the controls, the low-dose and the mid-dose groups.

Urination:The high dose group had fewer urine pools than the mid-dose group, the Na-citrate group had more urine pools than the low-dose group and fewer urine pools than the mid-dose group.

Foot-splay:The low-dose group showed sig. greater foot-splay than the mid-dose group and the Na-citrate group.

Forelimb grip strength:p-value not provided. Censored data analysis was required. Test results provided do not indicate the direction of the effects. The high dose was sig. different from the mid dose group (p<0.0001), the low-dose group (p<0.0001) and the controls (p<0.0001). The mid-dose group was sig. different from the low-dose group (p=0.0015) and the controls (p=0.0156). The Na-citrate group was sig. different from the controls (p=0.0242), the low dose group (p=0.0027), and the high dose group (p<0.0001).

Hind-limb grip strength:p-value not provided. Censored data analysis was required. The high dose was sig. different from the mid dose group (p<0.0001), the low-dose group (p<0.0001) and the controls (p<0.0001). The mid-dose group was sig. different from the low-dose group (p=0.0090) and the controls (p=0.0002). The Na-citrate group was sig. different from the controls (p<0.0001), the low dose group (p=0.0018), and the high dose group (p<0.0001).

Overall, the data provide little evidence for an Al effect on the autonomic function domain, the sensimotor function domain, or excitability. Significant wasting (physiological domain), was observed in the high dose females and appears related to treatment. In addition, there was limited evidence of effects on activity/well-being of the pups at the high dose reflected in fur appearance, deposits and rearing. There was some evidence of dose-response relationships between neuromuscular measurements – hind-limb and fore-limb grip strength - and Al-treatment in both males and females, although some of this effect may be secondary to body weight changes. Grip strength measurements showed considerably variability and a consistent ordering of the Al-treatment group responses (dose-response) was not observed at all time points.

The study report indicates that the grip strength equipment used had a maximum capacity of 700g. The number of determinations exceeding 700 g was reported to be 2-3% of the total number of measurements. Censored data analysis was also used to compensate for the cap to the maximum value. The report authors consider the 700 g capacity of the equipment not to have affected the results substantially. This is supported by the detection of a significant effect of treatment group.

Motor Activity:

Day 23 cohort, females:At PND 15, interval 11, the group effect was marginally significant (p=0.0435). The Na-citrate group had significantly higher ambulatory counts than the low-dose group (p=0.0214). At PND 17 and 21 there were no significant group effects.

Day 23 cohort, males:At PND 15, interval 7, the group effect was marginally significant (p=0.0465). The Na-citrate group had significantly higher ambulatory counts than the low-dose group (p=0.0462). At PND 17, a significant effect of group was observed at interval 2 (p=0.0316) but no (multiple-testing adjusted) pair-wise comparisons reached statistical significance. At PND 21, significant group effects were observed at intervals 2, 10, 11 and 12. At intervals 10, 11 and 12, the Na-citrate group mean ambulatory count was significantly greater than in the low and/or mid-dose groups. At interval 2, the control group exhibited a mean ambulatory count significantly greater than the mid-dose group.

No significant differences were observed among the female pups tested at PND 15, 17 and 21 with respect to mean ambulatory counts. Among male pups, however, significant group effects were observed on PND 17 and 21 due to significantly higher ambulatory counts among the Na-citrate animals compared to the mid-dose group.

Day 64 cohort, females:No significant group effect was observed at any interval or overall.

Day 64 cohort, males:Significant group effects were found at:

interval 5, p=0.0044 (high dose group sig. less than low dose group and controls);

interval 6, p=0.0319 (high dose group sig. less than mid-dose group and controls);

interval 7, p=0.0001 (high dose group sig. less than all other groups);

interval 9, p=0.0459 (high dose group sig. less than control);

interval 11, p=0.0088 (high dose group sig. less than controls, low dose and mid-dose group).

Day 120 cohort, females:A significant effect of group was observed at interval 6, p=0.0189 (low dose group sig. less than controls and high dose group). Overall, the repeated measures ANOVA showed a significant effect of group (p=0.0062). Pair-wise comparisons showed that the mean ambulatory counts in the low dose group were significantly less than in the high dose group, the controls and the Na-citrate group.

Day 120 cohort, males:A significant effect of group was observed at interval 3, p=0.009 (control group sig. less than mid-dose group and Na-citrate group). Overall, the effect of group was not significant.

Day 364 cohort, females/males:No significant group effect was observed at any interval.

No consistent pattern of group differences was observed in ambulatory counts across the different cohorts and intervals. The effects seen in the Day 64 cohort of males were not observed in the other cohorts.

Auditory Startle Response:

In general, the startle response data showed high variability with standard deviations close to mean response maximums. Mean response maxima decreased with block, consistent with habituation.

Day 23 cohort, females/males:The group effect was not significant.

Day 64 cohort, females:The group effect was significant (p<0.0001). Pair-wise comparisons did not show a pattern consistent with an Al-associated effect.

Day 64 cohort, males:The group effect was significant (p<0.0001). The high dose group was sig. less than the control but the low dose group was sig. greater than the control.

Day 120 cohort, females:The group effect was significant (p<0.0001). The Na-citrate group showed a sig. greater response than all the other groups.

Day 120 cohort, males:The group effect was significant (p<0.0001). The Na-citrate group was sig. greater than the low-dose group and the mid-dose group.

Day 364 cohort, females:The group effect was significant (p=0.01). The Na-citrate group was sig. less than the low-dose group and the mid-dose group.

Day 364 cohort, males:The group effect was not significant.

Overall, there was no consistent pattern suggesting an Al-treatment related effect on auditory startle.

T-maze:

The T-maze testing was conducted at PND 21.

Frequency of Alternation (visits to previously blocked arm as a percentage of all visits) areprovided below:

Group

Male

Female

Control

42.11

26.32

Low-Dose

25.00

42.11

Mid-Dose

31.58

47.37

High-Dose

63.16

31.25

Na-citrate

26.32

50.00

The effect of group was not significant (p=0.0866 in males, p=0.5529 in females.) As discussed by the study authors, the rates of alternation in the study were low, consistent with young animals that explore cautiously. The authors question the utility of these results based on the age of the animals being lower than ideal for the test.

Morris Water Maze:

Training Trial Latencies:There were no significant effects of treatment group in males or females for the Day 64 cohorts, the Day 120 cohorts or the Day 364 cohorts.

Platform-Removed Probe Test Search Strategies/Platform Visible Latencies/Platform Visible Type of Search:No significant treatment group effects in either sex or any of the cohorts.

Haematology:

Day 23 cohort:

Overall, haematological changes in the Day 23 cohort males and females were not considered clinically significant.

Day 64 cohort:

Both females and males in the high dose group showed low grade microcytic anaemia. In general, high dose animals had lower haematocrit, lower haemoglobin, lower mean cell haemoglobin, and lower mean cell volume but higher white blood cell counts than the other Al-treated groups.

Day 120 cohort, females:Absolute levels of granulocytes and agranulocytes were significantly elevated in the high dose group relative to the control, low- and mid-dose groups. MCH was significantly lower in the high dose group than in the control, mid-dose, and Na-citrate groups. Similar to the Day 64 cohort results, the MCV was significantly lower in the high dose group than in all other treatment groups also. The white blood cell count was significantly higher in the high dose group compared to that in the control,

the low-dose and the mid-dose groups.

Day 120 cohort, males:High dose males had been euthanized at this point.

The only significant inter-group difference was for MCV. Levels were significantly lower in the Na-citrate group than in the controls (p=0.0260).

Day 364 cohort, females/males:No significant effects of group. The anaemia had resolved in the females.

Coagulation parameters:

No significant treatment group effects were found for the coagulation parameters.

 

Applicant's summary and conclusion

Conclusions:
The results from this study are informative for developmental and neurotoxic effects due to prenatal and chronic postnatal exposure of rats to high doses of aluminium (30 mg Al/kg bw/day, 100 mg Al/kg bw/day and 300 mg Al/kg bw/day). As the F1 generation was dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. This does not affect the formal reliability of the study.

The results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. An effect of Na-citrate was observed in the female pups. Urinary tract pathology was observed in high dose rats, more frequently in the males. The results showed no evidence of an effect on memory or learning. Fairly consistent results were observed for the critical effect, fore- and hind-limb grip strength, and this was supported by the following less consistently observed effects also observed in the mid-dose (100 mg Al/kg bw/day) group: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools that controls); tail pinch (mid-dose females displayed more exaggerated responses); foot splay (mid-dose females had significantly narrower foot splay than the controls); the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls). No treatment-related differences in FOB characteristics were observed in the neonatal and juvenile pups. A LOAEL of 100 mg Al/kg bw/day for aluminium repeated dose toxicity is assigned based on this study.

Delayed sexual maturation, measured as delayed vaginal opening in females and delayed preputial separation in males, was observed in the high dose Al-citrate group of this study. The same effect, although somewhat less pronounced, was also seen in the sodium citrate control group. Based on the observed upward deviations from the target dose in the Al citrate groups and the data on water consumption seen in the first weeks after weaning, it is possible that both in the pre- and post-weaning stage, the animals in the Al citrate groups received considerably more citrate than the sodium citrate control group. Moreover, the calculated Al dose during the immediate post-weaning period was more than twice the target dose, which may have contributed to post-natal systemic toxicity due to exposure to the test substance. Given that effects were seen in both the Al-citrate high-dose group and the NA-citrate group, no Al-based LOAEL/NOAEL can be suggested based on the sexual maturation results in this study.

Body weight differences at end-of-weaning, relative to controls, occurred in the high-dose Al-citrate group as well as in the sodium citrate group and are considered to be treatment-related but the role of Al is unclear. The relative differences between the high-dose Al-citrate group and the sodium citrate group may be related to differences in liquid consumption.
Executive summary:

A double-blinded study was conducted to evaluate the developmental and neurobehavioural toxicological effects of orally administered aluminum (Al) citrate in rats in utero through to 1 year of age. 180 pairs of male and female Sprague-Dawley rats were bred and randomly assigned to one of five treatment groups: low, mid and high dose Al citrate, and water and sodium (Na) citrate control groups. The Al citrate dosing solutions were administered in the drinking water of pregnant dams to deliver target dosages of 30, 100 and 300 mg/kg body weight elemental Al. The Na citrate concentration was the molar equivalent of the high dose Al

citrate solution. The dose solution concentration of elemental Al for each treatment group was kept constant throughout the study and the dose received depended upon an expected water intake of 120 mL/kg/day. The highest concentration was very close to the solubility limit of Al citrate. Actual dose received depended on dose solution concentration and water consumption.

After delivery, litters were culled to 4 males and 4 females each, and 20 litters were randomly selected and retained from the available total number of litters per treatment group. From each culled litter, one male and one female were randomly assigned to one of 4 milestone groups designated by age at sacrifice to undergo neurobehavioural testing, neuro-histopathological examination and tissue metal analysis: postnatal days 23 (immediately post-weaning), 64, 120 and 364. Weaned rat pups were administered the

same treatments as the dams until sacrifice.

Actual dosages for dams were near or above target. Dams were observed, weighed and fluid consumption was measured on a regular basis. For neurobehavioral assessment, FOB (functional observational battery) assessments were conducted approximately weekly. The results indicated that Al at all dosage levels had little effect on the dams.

For the rat pups, the actual dosages were one third to one half of target for most of the 1 year lifespan, due to lower than expected fluid consumption. Pups were observed daily, weighed biweekly, and fluid consumption was measured weekly.

Neurobehavioral assessments on the pups included the following: FOB assessments were conducted on all pups several times before weaning and biweekly on the Sacrifice Day 364 group from weaning until the end of the study. Motor activity was measured periodically before weaning on the Sacrifice Day 23 group and shortly before sacrifice in the Day 64, 120 and 364 groups. Startle response was measured on all groups shortly before sacrifice. A T-maze test was conducted on the Day 23 group, while Morris water maze tests were conducted in the Day 64, 120 and 364 groups a few days before sacrifice.

At each sacrifice milestone, half the scheduled animals underwent perfusion fixation and neurohistopathological assessment, and the other half underwent a regular necropsy followed by brain weight measurement, clinical chemistry, hematology, and collection of tissues and blood for measurement of aluminum and other metals including copper, iron, zinc and manganese.

Adverse effects were most evident in the high dose offspring, but effects of Al were also detected at the mid dose.

In the neurobehavioral tests, the strongest effect was in the neuromuscular domain, with dose-related reductions in fore- and hind-limb grip strength and in the females, foot splay, because they were detected at the mid dose level as well as at the high dose. More subtle and inconsistent observations included, heightened FOB excitability in the females, slightly reduced and transient startle response and mild, slightly reduced motor activity. These observations provide weak evidence of Al effects because they were observed in one sex or in one sacrifice day group at a time. There was also no evidence of Al-related effects on learning/memory based on maze and habituation tests. There was no evidence of Al-related pathology beyond the urinary and gastrointestinal tracts, with no Al-related neuropathology observed.

Beyond the neurotoxicity detected, in mainly the high dose male pups, urinary calculi caused urinary tract pathology and all high dose male pups were euthanized at about three months of age. In both sexes, the high dose offspring exhibited lower growth rate and body weights, diarrhea, bloating, poor coats, hematuria, and delayed sexual development. However, some of these effects were observed in the Na citrate group as well. For clinical chemistry and hematology effects, the high dose group also exhibited microcytic anemia, elevated alkaline phosphatase, elevated serum calcium and urea, and lower total protein, albumin and globulin, relative to other groups. These symptoms are consistent with Al toxicity.

Concentrations of Al were highest by far in the blood, followed by bone (femur), liver and CNS tissues. For all sacrifice day groups, aluminum concentrations in the blood, liver and bone of both sexes were significantly higher in the high, and in some cases the mid dose groups relative to controls, peaking in concentration at Day 64 group (i.e., postnatal day 64). Also, in all sacrifice day groups, aluminum concentrations in blood, liver, bone and cortex (brain) were well correlated. Aluminum concentrations in the bone demonstrated the strongest correlation with dosage, with low, mid and especially high dose groups being significantly higher than Na citrate and controls for most sacrifice day groups/sexes. In the CNS tissues, concentration of aluminum was greatest in brainstem, followed by spinal cord, cerebellum and cortex. In general, aluminum concentration in CNS tissues was poorly correlated with dosage, with the exception of cortex where there were significant differences between high dose and control animals in several sacrifice day groups. Regional differences in aluminum concentration were most apparent in Day 23 and 64 groups, and only the cortex demonstrated an accumulation of aluminum with time.

In conclusion, in offspring exposed in utero through to one year of life, neuromuscular effects (based on the reduction fore- and hindlimb grip strength and foot splay) were detected at both the high (300 mg Al per kg, target) and mid (100 mg Al per kg, target) dosage levels. However, these effects are attributable to the lower body weights of the individual rats in this test. The individual data are not available, and neither are historical control data. Therefore, the neuromuscular effects cannot unequivocally ascribed to treatment with Al3+. The NOAEL was set based on this effect as 30 mgAl/kg bw in a conservative approach, forming the basis for the assignment of 100 mg Al/kg Body weight as the Lowest Observed Adverse Effect Level (LOAEL) for this study. The high dosage also produced physiological (urinary tract, growth rate, maturation rate, hematological and clinical chemical) adverse effects and concentrations of Al in blood, femur, liver and some CNS tissues were significantly higher than controls. However, there was no evidence of an effect on learning and memory in this study.