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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
Study period:
2008-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD 426 and OECD 452
Deviations:
yes
Remarks:
: food consumption was not studied; exposure during in utero (GD 6-21) and weaning period (post-natal day (PND) 1-21), but the exposure of the rats to Al citrate continued beyond this period, until 12 months of age in one cohort
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 TemperatureStability and homogeneity of dosing solutions under test conditions were established in a separate report (Appendix F). Reported stability - 21 days in the solutions. Sodium Citrate (Na citrate)- 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 and environmental conditions:
TEST ANIMALS- Source: Charles River Canada Inc.- Age at study initiation: 14 - 16 weeks at breeding- Weight at study initiation: Females: 242.5 - 333.4 g (target 160-360 grams); Males: 335.4 - 470.8 g (target 245-585 grams). - Housing: With the exception of the breeding period and dams with litters, animals were housed individually.- Caging: - Before the 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 also when hematuria or diarrhea was observed, or other issues as specified by the veterinarian. - All animals received plastic enrichment tubes only for environmental enrichment.- Diet: I. Diet: 5K75 irradiated rat chow until arrival of custom diet, thenII. 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 (Final Report/Draft, 2009, p.6).- Water: deionized H2O, ad libitum. Water levels of aluminium ranged from <1 – 160 ppb or 1 ug Al/L- 160 ug Al/L.- Acclimation period: 9 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18 - 26°C- Humidity (%): 30 - 70%- Air changes (per hr): ≥ 10 per hour in the roomand within ventilated cages, animals were expected to experience approximately 50 air changes per hour using room air for both intake and exhaust. - Photoperiod (hrs dark / hrs light): ~12 hr. lightADDITIONAL INFORMATION- Number of Animals in the Study: The study began with 180 pairs to ensure 100 litters of progeny were delivered within the allotted timeframe (5 consecutive days);

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of Al concentrations in the formulations and dosing solutions The formulations and dosing solutions were prepared based on the Al content specified in the supplier’s Certificate of Analysis. Samples of at least 5 mL of each dose level of the dosing solution and also for the sodium citrate reference solution were stored and transported (overnight; ambient temperatures) then analyzed for aluminium content by ICPMS. Samples were collected from the first formulation, then from each week’s formulation for 4 weeks, then at 4 week intervals and, at the last dose preparation, until the end of the study.The analyses showed that the dosing solutions prepared from the third lot of Al citrate had unexpectedly low Al concentrations, about 25% below target. The amount of Al citrate was thus increased to compensate. The Certificate of Analysis from the supplier gave a nominal concentration of 8.7% Al for this lot of the test item. The lower than specified Al levels (6.6% by analysis) were later confirmed by the supplier. The Al concentrations in the dosing solutions differed from target by -30% to +39% throughout the study (Appendix B, Table B26. Dose Verification). The stability and homogeneity of the dosing solutions under test conditions were determined in a separate study (Appendix F: Evaluation of the Stability and Homogeneity of Aluminium Citrate in Sodium Citrate and Aluminium Citrate Dosing Solutions by ICP-MS; ALB206-070144-5120). The results indicated that aluminium concentrations (at 2.5 g/L Al-citrate or endogenous Al levels in 27.2 g/L sodium citrate) remained stable and well-mixed in aqueous solution in a feeding bottle at room temperature for a 21 day period.Aluminium Levels in the Diet and Vehicle Samples of the different diets were analysed for aluminium, iron, manganese, copper, and zinc. For the enriched Purina AIN-93G, one sample was collected prior to the study and another was collected 6 weeks after the experimental starting date. One sample of Purina AIN-93M was taken prior to the switch in diets and another 6 weeks later. When new lots of the maintenance diet were received, they were tested before entering the study and again 6 weeks after being introduced. Levels of aluminium in the diets were 6-9 ppm (6-9 µg/kg diet) over the study.Levels of aluminium in the Nanopure water ranged from <1 – 160 ppb or 1 ug Al/L- 160 ug Al/LAluminium levels in the Reference ItemAluminium levels were also determined similarly in the sodium citrate solutions. Dose verification analyses showed levels from 40-249 µg Al/L (with 6 of 19 measurements ≥100 µg Al/L; see Appendix B, Table B26).All analyses were appropriately blinded.
Details on mating procedure:
Sires and dams were allocated into breeding pairs by using SAS PROC PLAN procedure. Animals 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.DamsGD 6 to PND 21.Pups (males and females)PND 22 to PND 364.Cohort 1 – GD 6-21, PND 1-22 Cohort 2 – GD 6-21, PND 1-64Cohort 3 – GD 6-21, PND 1-120Cohort 4 – GD 6-21, PND 1-364
Frequency of treatment:
ad libitum (daily, 7 days per week.)
Duration of test:
1 year
Doses / concentrations
Remarks:
Doses / Concentrations:3225 mg/Al citrate/ kg bw/day (Group E); 1075 mg /Al citrate/kg bw/day (Group D); 322.5 mg/Al citrate/kg bw/day(Group A); Control: 0 mg/Al citrate/ kg bw/d– (Group C); Sodium citrate dihydrate (Na citrate group) (Group B). Basis:other: Other: nominal in water; 300 mg Al/kg bw/day (Group E); 100 mg Al/kg bw/day (Group D); 30 mg Al/kg bw/day(Group A); Control: deionised water – (Group C); Sodium citrate dihydrate (Na citrate group) (Group B).
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:
other: A control group received water only. A citrate control group received sodium citrate at dose equimolar to citrate in the aluminium High dose group/300 mg Al/kg/Group E - 27.2 g/L.
Details on study design:
Dose SelectionDoses were selected based on the results of a previous study, TEH-104 (Aluminium citrate: A 90 day toxicity study in rats. 2008. ToxTest, Alberta Research Council, Report No.: TEH-104) and the maximum solubility of aluminium citrate in water (high dose). The number of dose levels and dose spacing was according to guideline.Dams & SiresAllocation to Treatment Groups Rats were randomly allocated to treatment groups and randomly selected for breeding using the SAS PROC PLAN procedure.Allocation to Shelf/RackPrior to breeding, a Youden square was used to produce equal representation of the treatment groups within each shelf of the rack. Location of the breeding pairs was also dictated using a Youden square. After insemination, each dam was returned to her original cage and remained at that location until postnatal day 1 or euthanasia.As the proportion of dams in each treatment groups that would deliver on a specific day could not be predicted, extra breeding pairs were included in the study. After the end of the week during which deliveries were expected, litters that were eligible to enter the study (≥4 pups of each sex) were randomly chosen to provide a balanced distribution of litters per treatment group per delivery day. PupsLitter NormalisationAt PND 4, litters were normalized to 4 males and 4 females using random numbers. Of the extra pups, 4 males and 4 females per treatment group were randomly chosen for whole body aluminium, iron, manganese, copper and zinc assay. Allocation to CohortAlso 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. Allocation to Shelf/RackPups were weaned at PND 22 by moving them to individual ventilated caging using another Youden square to determine their distribution within the rack.BlindingAssessors 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). Dams and sires were identified by ear tags 3 days after arrival at the facility. Pups were identified on PND 4 within micro tattoo on the feet, and on PND 21 (at weaning) with an ear tag. Cages were identified by cage cards.

Examinations

Maternal examinations:
Parental animalsDams Morbidity and MortalityAll dams underwent daily morbidity and mortality checks and a clinical examination was performed on the day of delivery.Functional Observational Battery (FOB)Schedule: Gestational days (GD) 7 and 13 and on postnatal days (PND) 3 and 10. Content: The FOB (adults) included: - cage-side assessment, - handling assessment, - open field observations (posture, involuntary movements, abnormal motor movements), and - sensory and neuromuscular observations: - footsplay and - fore-limb grip strength and - hind-limb grip strength. Body weightsSchedule: GD 6, 13, and 20, PND 1, 8, 15, and 22. Body weight on PND 1 was examined but not included in the analysis. Water consumptionSchedule: GD 6, 13, 20, and then on PND 1, 8, 15, and 22.
Ovaries and uterine content:
No data.
Fetal examinations:
Not applicable.
Statistics:
See "any other information on materials and methods incl. tables"
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 effectsDetails on maternal toxic effects:MortalityNo mortality was observed in the dams during the gestation and postnatal periods in the control group, the low-dose group, the mid-dose group or the high-dose group; 20 dams were euthanized on the scheduled dates in each group. One dam that stopped nursing was euthanized early in the sodium citrate group. Body weightThe ANOVA showed a significant effect of group (p=0.021). This was due to lower body weights in the sodium citrate group. At PND15, the mean weight of the Na-citrate group was 7.3% less than in the controls. There were no significant differences in mean body weights in dams between the aluminium-treated groups and the control group during the gestational and postnatal period.Gestation LengthThere were no statistically significant differences in gestational length between the different treatment groups.Clinical ObservationsAll dams underwent daily morbidity and mortality checks during the gestational period and a clinical examination was performed on the day of delivery. Abnormal clinical observations were reported for only one dam during the gestational period.During the postnatal period, 4 animals in the control group, 8 in the Na-citrate group, 4 in the low-dose group, 6 in the mid-dose group, and 12 in the high dose group exhibited clinical signs. Most signs were considered mild, for example alopecia and porphyrin staining. Slight dehydration was noted in 4 dams in the Na-citrate group. Diarrhoea was reported in 8 dams in the high dose aluminium group only, and thus appears to be a treatment-related effect.Water ConsumptionThe table below the ranges of mean fluid consumption in mL/day (mL/kg bw/day) for the different groups for the gestation and lactation periods:Group/PeriodGestation LactationControl23.0 to 31.5 (67 to 79)35.1 to 60.6 (99 to 179)Low Dose35.9 to 43.7 (103 to 108)40.1 to 60.9 (114 to 177)Mid-Dose42.0 to 45.2 (112 to 123)40.9 to 69.0 (136 to 201)High-Dose27.4 to 31.3 (78 to 80)39.7 to 70.2 (120 to 211)Na-citrate26.2 to 29.3 (66 to 76)35.1 to 68.0 (106 to 213)A significant effect of group was found in the ANOVA (p<0.0001). Pairwise between-group comparisons showed that the low dose group consumed significantly more water than the sodium citrate (p=0.011) and water control (p=0.0028) groups. The mid-dose group consumed significantly more than the sodium citrate (p<0.0001), water control (p<0.0001) and high dose groups (p=0.023). The differences were most marked during the gestation period. As increased water consumption was not observed in the high dose group, the effect is not likely due to treatment.Daily Al dosageThe 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. Despite the deviations from the target dose, the low, medium and high dose groups showed the required trend of lowest to highest maintaining group differences in dosage.FOBDuring the gestation period, approach response, arousal, bizarre behaviour, circling, clonic convulsions, clonic convulsions rating, gait, posture, pupil response, pupil size, startle, stereotypic behaviour, tail pinch, tonic convulsions, tonic convulsions rating, total gait, tremors, tremors rating, vocalization, and writhing were zero for all dams.The group effect (repeated measures ANOVA) for defecation (p=0.052), rearing (p=0.344), urination (p=0.487) and foot splay (p=0.089) did not reach statistical significance. A significant group effect was observed for hind limb grip strength (p=0.0047; censored analysis) driven by a lower grip strength in the Na-citrate group compared to the low and high dose groups. During the postnatal period, bizarre behavior, circling, clonic convulsions, clonic convulsions rating, gait, posture, pupil response, stereotypic behavior, tonic convulsions, tonic convulsions rating, total gait, tremors, tremors rating, and writhing were zero for all dams. The group effect (repeated measures ANOVA) for approach response (p=0.518), arousal (p=0.146), defecation (p=0.096), pupil size (p=0.413), rearing (p=0.151), startle (p=0.668), tail pinch (p=0.242), urination (p=0.793), vocalization (p=0.092), and foot splay (p=0.142) did not reach statistical significance. A significant across groups difference (censored analysis) was observed for forelimb grip strength (p=0.0031). Pair-wise comparisons showed that the mid-dose group was significantly less than the sodium citrate group (p=0.0005) and the high dose group (p=0.0115). The low dose group was significantly less than the sodium citrate group (p=0.012) and the control group was significantly less than the sodium citrate group (p=0.0076). The group effect for hind limb grip strength did not reach statistical significance (p=0.073) so pair-wise comparisons were not conducted.Overall, there was no consistent effect of treatment group on any of the FOB characteristics in the dams.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Basis for effect level:
other: no adverse effects at highest dose tested

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examinedDetails on embryotoxic / teratogenic effects:Not applicable.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: urinary tract pathology, hindlimb grip strength, forelimb grip strength
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: urinary tract pathology, hindlimb grip strength, forelimb grip strength

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Offspring results:

Mortality

Mortalities/unscheduled euthanizations observed in each group (extracted from Appendix B, Table B8).

 

Female

Male

 

Died

Euthanized

Died

Euthanized

Control

4

4

3

1

Low-Dose

1

1

2

3

Mid-Dose

0

0

2

0

High-Dose

4

9

8

37

Na-citrate

3

2

7

3

Note: Pups that were euthanized because their dam stopped nursing were not included in these numbers. Pups that were switched and data excluded from the study were also not included.

The main cause of mortality and the reason for the high number of euthanizations in the high dose group was urinary tract pathology (see Pathology results for more detail) – hydronephrosis, ureteral dilation, obstruction and/or presence of calculi.

Clinical Observations

In the Day 23 cohort: the only clinical observations noted were in the high dose animals - abdominal distention (n=2; 1 female, 1 male), and small and cold animals (n=3; 1 female, 2 males). No treatment-related effects were evident.

In the Day 64 cohort: 1 female in the control group was thin and showed abdominal distention and 3 males in the Na-citrate group were thin and had poor coats. In the high dose group, 1 female and 7 males had diarrhea, poor coats and were slightly dehydrated, an effect likely due to treatment.

In the Day 120 cohort: No abnormal observations were noted for the control, low or mid-dose groups. 2 females and 1 male were thin with poor coats in the Na-citrate group. In the high dose groups, 5 females and 10 males had diarrhoea, 1 female had haematuria with the diarrhoea. Enlarged kidneys were noted in three animals.

In the Day 364 cohort: haematuria was observed in 1 female in the high dose group, 1 female in the control group, and 2 females and 6 males in the Na-citrate group. Note: After about half of the high dose males died from urinary tract blockage or were euthanized on the basis of the severity of the clinical signs relating to urinary tract pathology, the remaining high dose males were euthanized.

Masses and skin lesions and abnormalities were observed but did not appear to be related to treatment. Seizures were observed in 2 high dose females, 2 mid-dose males and 2 mid-dose females, 1 female in the Na-citrate group and 1 control female. The incidence of seizures does not appear related to treatment. Limping noticed in Day 364 cohort animals was not associated with treatment and likely resulted from multiple foot splay assessments.

In summary, clinical observations that were found associated with treatment, either directly or secondary to renal failure, were poor coat, weight loss, diarrhea, and haematuria. Considering the animals dosed with Al-citrate, these signs were only found in the high dose group and were more frequent in males. Haematuria was also observed in the Na-citrate group in the Day 364 cohort.

Body Weight

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:

Analyses for the individual cohorts sacrificed in the post-weaning phase were provided in Appendix E (Statistician’s Report) accompanying the final report. The final report itself focused on interpretation of the data from the Day 364 cohort as it covered the full duration of the study.

Day 23 cohort, females: Na-citrate group animals were significantly lighter than the low dose (p=0.0348) and the control group (p=0.0305) animals.

Day 23 cohort, males: Na-citrate group animals were significantly lighter than the low dose (p=0.0014) and the control group (p=0.0033) animals.

Day 64 cohort, females: High dose females 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: High dose males were significantly lighter than all the other dose groups. 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.

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.

Growth Curve Parameters

In female pups, there was a significant effect of group on asymptotic weight (p<0.0001), days to 50% final body weight (bw) (p=0.0002) and growth rate (p<0.0001). Pair-wise comparisons showed that the high dose group had significantly lower mean asymptotic weights than the control and mid-dose groups (p=0.0009 and p=0.0081, respectively). Days to 50% bw and growth rate were significantly lower in the high dose compared to the control. The mean asymptotic weight in the Na-citrate group was significantly lower than in both the control and mid-dose groups.

In male pups, when data after day 84 were excluded, asymptotic weight and days to 50% bw were significantly lower in the high dose group than in the other treatment groups. Treatment group did not show a significant effect on growth rate, however (p=0.0729) [data from Statistical Report, Table 5.15]. When high dose males were excluded from the analyses, there was no significant group effect on any of the growth curve parameters (reported qualitatively in the Final Report).

The inclusion of six erroneous body weights had no effect on the interpretation of the results.

Water 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.

Actual Doses Received

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.

Organ Weight

Brain weights.

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 (data extracted from Table 4 of the final report):

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.

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 pups)

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.

FOB (juveniles)

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.

Overall, no Al-citrate related treatment effects were observed in the FOB observations.

FOB (adult pups)

Day 364 cohort

Females

Normal observations were found in all females for tonic convulsions (home cage), clonic convulsions (home cage), tremors (home cage and open field), posture (home cage and open field), conjunctivitis (handling observations), and total gait (open field). Although some non-normal observations were reported, there were no significant group differences for palpebral closure, lacrimation, red crusty deposits (eye), ocular exudates, exophthalmus, muscle tone, piloerection, ease of handling, ease of removal, vocalizations, gait, stereotypic behaviour, bizarre behaviour, circling, tonic convulsions (open field), clonic convulsions (open field), approach response, startle response and writhing. Significant group differences were observed for:

FOB Parameter Group effect Pairwise Differences

Wasting P=0.0040 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).

- treatment-related effect

Fur appearance P=0.0001 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 P<0.0002 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. Not consistent with a treatment-related effect.

Eye opacity P=0.0001 The low dose had sig. more than the other groups. Not treatment-related.

Salivation P=0.0230 Low and mid-dose had sig. more salivation than the high dose group and the controls. Not consistent with a treatment-related effect.

Arousal (open field) P=0.0011 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 P<0.0001 The high and mid-dose groups have more faecal boluses than the low-dose group, the controls and also the Na-citrate group. Likely a treatment-related effect.

Defecation characteristics P<0.0001 As above

Pupil response P<0.0001 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 P=0.033 The Na-citrate group is sig. more abnormal than the high dose group, the controls and the mid-dose group. Not consistent with an Al-treatment-related effect.

Rearing P<0.0001 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. Not consistent with a treatment-related effect.

Tail pinch P=0.0001 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. Overall, not clearly consistent with a treatment-related effect.

Urination P=0.0001 The Al-treated groups and the controls had sig. more urine pools than the Na-citrate group.

Urine characteristics P=0.0099 The low-dose, mid-dose and controls had sig. more urine pools and abnormal colour than the Na-citrate group.

Foot-splay P<0.0001 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. Weak evidence of dose-response and a treatment-related effect.

Forelimb grip strength P<0.0001 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). Some evidence of dose-response; treatment-related effect.

Hind-limb grip strength P<0.0001 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). Some evidence of dose response; treatment related effect.

Males

Normal observations were found in all males for tonic convulsions (home cage and open field), clonic convulsions (home cage and open field), tremors (home cage and open field), posture (home cage and open field), conjunctivitis (handling observations), ocular exudates (handling observations) and writhing (handling observations). Although some non-normal observations were reported, there were no significant group differences for wasting, lacrimation, muscle tone, salivation, ease of handling, ease of removal, arousal, total gait, stereotypic behaviour, circling, pupil response, pupil size, startle response, and approach response. Significant group differences were observed for: fur appearance, mouth and nose deposits, eye opacity, red crusty deposits, exopthalmus, piloerection, defecation, defecation characteristics, tail pinch, rearing, urination, urine characteristics, foot splay, forelimb grip strength and hind-limb grip strength. Vocalizations, gait and bizarre behaviour were not analyzed due to skewed distributions and missing data.

FOB Parameter Group effect Pairwise Differences

Fur appearance P<0.0001 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 P=0.0216 High-dose group had sig. more deposits than the low-dose group and the mid-dose group.

Eye opacity P<0.0001 Low-dose group had sig. more loss than controls, the mid-dose group and the Na-citrate group. Not consistent with a treatment-related effect.

Red Crusty deposits P=0.0087 The mid-dose group had sig. more deposition than the controls and the Na-citrate group.

Exophthalmus P=0.0064 High dose group had sig. more eye bulging than the controls, the mid-dose group, and the Na-citrate group.

Piloerection P=0.0015 The mid-dose group had sig. more piloerection than the controls, the low dose group and the Na-citrate group.

Defecation P<0.0001 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. Not consistent with a treatment-related effect.

Defecation characteristics P<0.0001 Not clearly related to treatment.

Rearing P<0.0001 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. Variable and not clearly consistent with a treatment-related effect.

Tail pinch P=0.003 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. Not consistent with a treatment-related effect.

Urination P<0.0001 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. Overall, not consistent with a treatment-related effect.

Urine characteristics P<0.0001 Not clearly related to treatment.

Foot-splay P=0.0004 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 then 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 then 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: No significant group effect was observed at any interval or overall.

Day 364 cohort, males: No significant group effect was observed at any interval. Although the group effect from the repeated measures ANOVA was significant (p=0.0088), all adjusted p-values from pair-wise comparisons were >0.05.

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: The group effect was not significant.

Day 23 cohort, 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) are provided 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

No significant treatment group effects in either sex or any of the cohorts.

Platform Visible Latencies

No significant treatment group effects in either sex or any of the cohorts.

Platform Visible Type of Search

No significant treatment group effects in either sex or any of the cohorts.

 

Overall, there was no evidence for effects of aluminium on animal performance in the Morris Water Maze Test.

Haematology

The following haematology parameters were assessed: absolute agranulocytes, absolute granulocytes, agranulocytes, granulocytes,

haematocrit, haemoglobin, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, platelet count, nucleated red blood cells, red blood cell count, and white blood cell count.

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: No significant effects of group.

The anaemia had resolved in the females.

Day 364 cohort, males: No significant effects of group.

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 citrate 3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 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. A LOAEL of 1075 mg AlCitrate/kg bw/day (100 mg Al/kg bw/day) for aluminium toxicity is assigned based on this study. 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 (1075 mg AlCitrate/kg bw/day; 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).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. As such, 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. No treatment-related differences in FOB characteristics were observed in the neonatal and juvenile pups.
Executive summary:

This study was designed “to develop data on the potential functional and morphological hazards to the nervous system that may arise from pre-and post-natal exposure to aluminium citrate”. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions of aluminium citrate at 3 dosage levels of aluminium citrate 3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day). Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al citrate and Na-citrate were administered to Dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.

 

Endpoints and observations in the dams included water consumption, body weight, a Functional Observational Battery (FOB), morbidity and mortality. Endpoints were assessed in both female and male pups that targeted behavioural ontogeny (motor activity, T-maze, auditory startle, the Functional Observational Battery (FOB) with domains targeting autonomic function, activity, neuromuscular function, sensimotor function, and physiological function), cognitive function (Morris swim maze), brain weight, clinical chemistry, haematology, tissue/blood levels of aluminium and neuropathology at the different dose levels and time points PND 23, 64, 120 and 364.

 

Statistical analyses were undertaken according to intention-to-treat, with appropriate consideration of multiple testing issues and, through the study design, also the unit of analysis. Censored analyses using survival analysis (Fixed Effects Partial Likelihood) were required for the grip strength measurements due to an equipment-defined maximum value. Females and males were analysed separately.

 

There were no significant Al-citrate treatment-related effects on mean body weights observed in the dams during the gestation and postnatal periods. The Na-citrate group, however, was significantly lighter than the control group on PND 15 (7.3%; p=0.0316). Eight dams in the high dose aluminium group were found to have diarrhoea compared with none in the other treatment groups. The low and mid-dose Al-citrate groups consumed more water than the control group but the high dose group did not, suggesting that the effect was not simply due to treatment. There were no significant treatment-related differences in gestational length. There were no consistent treatment-related effects observed for the tests in the dams. Due to the differences in water consumption, the % of target dose differed between groups and with time through the study. In the high dose group of dams, the actual dose during the first week of gestation was 200 mg Al/kg bw/day, 67% of the target dose (300 mg Al/kg bw/day). In the last week before weaning (and sacrifice), the actual dose received by the dams was close to 175% of the target dose. Statistical analyses comparing the actual doses received by the low, mid- and high- Al-citrate treatment groups showed that the order of the dose groups was maintained, however.

 

The most notable treatment-related effect observed in the offspring was renal pathology – hydronephrosis, ureteral dilation, obstruction and presence of calculi - most prominently in the male pups. Higher mortality and significant morbidity were observed in the male pups in the high dose group; leading to euthanization of this group atca. study day 89. Clinical observations that showed a relationship with treatment, either directly or secondary to renal failure, were poor coat, weight loss, and haematuria. Diarrhoea was also observed. These signs were found only in the high dose Al-citrate treatment group. Haematuria was also observed in some animals in the Na-citrate group in the Day 364 cohort. Dosing with Al-citrate was associated with a reduction in body weight. 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. In the Day 120 cohort male pups, the mid-dose animals were significantly lighter than the controls. An effect of Na-citrate was observed in the female pups in the Day 364 cohort. Overall, dosing of animals with aluminium citrate led to higher fluid consumption than in the control animals. Dosing with Na-citrate was associated with a significant increase in fluid consumption relative to that of 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. 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 dose levels. For most of the study period, the actual dose received was less than the target dose in all treatment groups.

 

In the female pups, 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 Al-citrate group, a significant difference (p<0.0001). In males, 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, also a significant difference in the pair-wise comparisons (p<0.0001). Delayed development of both male and female pups wasobserved in the high dose Al-citrate group and also in the Na-citrate group. The effect is considered treatment-related but whether the effect is secondary to decreases in body weight is not clear, however. In addition, as an effect was observed in the Na-citrate group, the role of aluminium in causing this effect can neither be concluded nor excluded.

FOB observations showed no clear treatment-related effect among the neonatal Day 364 cohort pups that were assessed at PND 5 and 11 or in the juvenile pups assessedca.PND 22. In the adult pups, 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. Characteristics of defecation (number of boluses) also showed differences with treatment. In addition, there was limited evidence of effects on activity/well-being of the pups at the high dose as reflected in fur appearance, deposits and rearing. There was some evidence for dose-response relationships between neuromuscular measurements – hind-limb and fore-limb grip strength and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. Although the endpoint most consistently associated with Al-citrate treatment, 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. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning Day 23 cohort) or the Morris Water Maze test (Day 120 cohort).

 

Haematology parameters showed no significant treatment-related effects in the Day 23 cohort. In the Day 64 cohort, however, both males and females showed low grade microcytic anaemia (significantly lower mean cell volume, mean cell haemoglobin, and haematocrit). The anaemia had resolved by the end of the study in the Day 364 cohort females. Clinical chemistry results showed serum chemistry changes associated with aluminium toxicity such as elevated alkaline phosphatase and serum calcium. The authors state the levels still remained within the normal range. Effects were most pronounced in the Day 64 cohort animals. By Day 364 in the females, alkaline phosphatase levels did not differ significantly between the treatment groups.

 

Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control groups. There were no significant sex differences. These results suggest transfer of Al from dams to pupsin utero, although a contribution from breast milk PND 0 to 4 is also possible. Concentrations of Al in bone showed the strongest association with Al dose and some evidence of accumulation over time in all of the Al-treated groups. Of the central nervous system tissues, Al levels were highest in the brainstem. Although levels of Al were relatively low in the cortex (< 1µg/g), they were positively associated with Al levels in the liver and femur.  In females, Al levels in the high dose group remained elevated relative to the other groups at all time points suggesting that accumulation might have occurred.

 

Pathological examinations showed clearly that urinary tract pathology was a treatment-related effect. The only other treatment-related effect reported on necropsy was watery, tan-coloured fluid in the digestive tract in some high dose animals, more frequently in the Day 64 group. None of the lesions seen on histopathological examination of brain tissues of the Day 364 group was treatment-related and, as these were also seen in the control group, were likely due to ageing.

 

This study has many strengths. It was conducted according to GLP with a design based on OECD TG #426. The study used adequate numbers of animals and randomization to reduce bias, assessed endpoints in both female and male offspring, and studied a wide range of neurotoxicity endpoints. Haematology, clinical chemistry, pathology and general toxicity endpoints were also assessed. Three dose levels were used although the highest was close to the.Although representative of actual human exposures, extending the period of exposure beyond weaning until day 364 leads to ambiguity in interpretation of the results as effects observed later in the study may have resulted from either later exposures or exposures during periods critical for development. There were a number of deviations from protocol that are clearly described in the study report. Overall, these deviations were unlikely to have impacted the results of the study.

 

The results from this study are informative for neurotoxic effects due to combined prenatal and chronic postnatal exposure of rats to high doses of aluminium citrate 3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day).As the offspring were dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. Urinary tract pathology was observed in rats in the high dose group, more frequently and more severe in the males. The study showed no evidence of an effect of Al-citrate on memory or learning but a more consistent effect was observed in endpoints in the neuromuscular domain.

 

The ambiguity as to the critical period of exposure and the time-varying water consumption complicate the derivation of a point-of-departure from this study. A LOAEL of 1075 mg AlCitrate/kg bw/day (100 mg Al/kg bw/day) for aluminium toxicity is assigned. The critical effect was a deficit in fore- and hind-limb grip strength in the mid-dose group, supported by evidence of dose response and less consistently observed effects in the mid-dose animals: 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 than controls); tail pinch (mid-dose females displayed more exaggerated responses); foot-splay (mid-dose females had significantly narrower foot-splay than the controls); and the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls).