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REACH

Reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate

EC number: 914-920-3 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

OECD 426 and 452, rat: NOAEL = 3225 mg/kg bw/day of the read across aluminium citrate;

similar to OECD 413, rat: LOAC=15 mg/m³ of the read across aluminium hydroxychloride

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:: chronic toxicity: oral
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: Comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:: reference to same study
Qualifier:: equivalent or similar to guideline
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
Principles of method if other than guideline:: The study design was developed based on guidelines “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” (Final Report).
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Canada Inc.
- Age at study initiation: 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).
- Fasting period before study:
- Housing & Caging: Except during the breeding period and when dams were with their litters, animals were housed individually. During the breeding period, sire/dam pairs were housed in wire bottomed cages to facilitate identification of vaginal plugs. Pregnant dams were housed in conventional shoebox caging during gestation and also during the lactation period with their pups. After weaning until PND 120, pups were housed individually in ventilated caging after which they were transferred to shoebox caging.
Standard corn cob bedding was used with the exception of the gestation and lactation periods and when haematuria, diarrhoea or issues specified by the veterinarian required other bedding. At these times Harlan TEK-Fresh diamond soft bedding was used.
Plastic environmental enrichment tubes were available for all animals.
- Diet: Animals were fed 5K75 irradiated rat chow until arrival of the custom diet. Starting at least five days prior to breeding, the animals were fed Purina AIN-93G-Irradiated, a growth/lactation diet. AIN-93G was fed to all animals until PND 95-99. After PND 95-99, the animals were switched to a maintenance diet, Purina AIN-93M – Irradiated, for the remainder of the study.
- Water: Deionised H2O (or the dosing solutions), ad libitum.
-Levels of Al were determined in both the diets and in the deionised water (reported below in the section on exposure).
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): ≥ 10 per hour in the room
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:: oral: drinking water
Vehicle:: water
Remarks:: Deionised water (same as water above). Supplier: The water is produced from the Nanopure II deionization systems installed within the test facility fed by the facility reverse osmosis water.
Details on oral exposure:: PREPARATION OF DOSING SOLUTIONS: Methods: 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.

The stability and homogeneity of the dosing solutions under test conditions were determined in a separate study. 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 mg/kg diet) over the study.

Levels of aluminium in the Nanopure water ranged from <1 – 160 ppb or 1 µg Al/L- 160 µg Al/L

Aluminium levels in the Reference Item
Aluminium 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).

All analyses were appropriately blinded.
Duration of treatment / exposure:: Dams
Day 6 of gestation – day 21 post-natal

Pups (males and females)
Day 22 – 364 post-natal
Cohort 1: Day 6 of gestation – day 21 post-natal, day 1-22 post-natal
Cohort 2 : Day 6 of gestation – day 21 post-natal, day 1-64 post-natal
Cohort 3: Day 6 of gestation – day 21 post-natal, day 1-120 post-natal
Cohort 4 : Day 6 of gestation – day 21 post-natal, day 1-364 post-natal
Frequency of treatment:: daily, 7 days per week
Remarks:: Doses / Concentrations:
Low dose group (Group A): 30 mg Al/kg bw; Mid dose group (Group D): 100 mg Al /kg bw; High dose group (Group E): 300 mg Al/kg bw; Control I: Distilled water; Control II: Na citrate group (Group B): 27.2 g/L citrate
Basis:
nominal in water
No. of animals per sex per dose:: 20 P females;
10-20 F1 females and 10-20 males
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 no treatment; other: 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:: Sires and dams were allocated into breeding pairs using the SAS PROC PLAN procedure.
Animals were allowed to breed for up to five consecutive nights. Female animals were checked daily for the presence of vaginal plugs. The date of breeding was defined as the day when a vaginal plug was first detected. Breeding pairs were then separated.

- Dose selection rationale: Doses 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 & Sires
Allocation to Treatment Groups
Rats were randomly allocated to treatment groups and randomly selected for breeding using the SAS PROC PLAN procedure.

Allocation to Shelf/Rack
Prior 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.

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.

Pups
Litter Normalisation
At 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 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.

Allocation to Shelf/Rack
Pups were weaned at PND 22 by moving them to individual ventilated caging using another Youden square to determine their distribution within the rack.

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). 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.
Observations and examinations performed and frequency:: DAMS
Morbidity and Mortality
All 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:
- foot splay and
- fore-limb grip strength and
- hind-limb grip strength.

Body weights
Schedule: 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 consumption
Schedule: GD 6, 13, 20, and then on PND 1, 8, 15, and 22.

PUPS
Body weights
Schedule: PND 1, 4 (prior to assignment to cohorts), 8, 11, 15, 17, 22, 29, and biweekly thereafter (with the exception that a 13-day interval was used between PND 43 and 56), and immediately prior to sacrifice.

Water consumption
Schedule: Cohorts 2 to 4 (Days 64, 120 and 364) on PND 22 and weekly thereafter until just prior to sacrifice. The pups in the Day 23 (Cohort 1, pre-weaning cohort) cohort had their own water bottles for one day after weaning and before sacrifice, but water consumption was not measured in these animals.
Developmental landmarks
Female pups were monitored for vaginal opening starting on PND 26.
Male pups were monitored for preputial separation starting on PND 35.

Blood collection
Selection of pups: Ten males and ten female pups from each treatment group were randomly selected for blood collection.
Methods: Terminal blood samples were taken from anesthetized animals on the day of scheduled sacrifice, prior to euthanasia. Venipuncture of the abdominal vena cava was used with the exception of Cohort 1(Day 23) animals which required cardiac puncture due to the small size of the rats.

Blood analysis - Clinical chemistry
In serum, alanine aminotransferase, albumin, albumin/globulin ratio, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatine kinase, creatinine, globulin, glucose, sorbitol dehydrogenase, phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea nitrogen were measured.

Haematology parameters
The following parameters were evaluated on an Abbott Cell-Dyn® 3700 CS using Abbott reagents:
- Red Blood Cell count and morphology
- White Blood Cell count
- Differentiation of Granulocytic and Agranulocytic White Blood Cell
- Haematocrit
- Haemoglobin
- Mean Cell Haemoglobin
- Mean Cell Volume
- Mean Cell Haemoglobin Concentration
- Platelet count.

Coagulation panel
Prothrombin time (PT) and partial thromboplastin time (PTT) were assessed using a Coagamate® XM with Somagen reagents.

Aluminium levels in blood
Blood samples (200 µL) of animals undergoing normal necropsy were taken into polypropylene containers and sent to the test site for analysis by ICP-MS.

Quality Control & Exclusion of samples
Samples with blood clots with largest dimension >2 mm were not run for haematology.

Samples obtained by cardiac puncture were included in analyses as long as sample quality was adequate, recognizing that samples collected by this method may contain artifactually high levels of creatine kinase and aspartate aminotransferase.

Most haemolysed samples of sufficient quality were included in clinical chemistry analyses. For all assays with the exception of aspartate aminotransferase, samples that were excluded exceeded the maximum allowable haemolysis index specified by the manufacturer of the reagents.

No specific neurobiochemical testing was performed

No ophthalmological examination was performed
Sacrifice and pathology:: Necropsy of Animals Undergoing Terminal Blood Collection/Analysis of Metal Levels in Tissues

Half of the animals scheduled to be sacrificed at the end of each observation period (10 males and 10 females per treatment group planned) were euthanized by exsanguinations under isoflurane anaesthesia and underwent a necropsy supervised by a Board-Certified Veterinary Pathologist. Animals that were found dead during the study also underwent a necropsy.

Brain weight
The brains of these animals were dissected and weighed. Brain weights were not recorded for rats that were found dead or were euthanized prior to the end of the study, including the culls.

Liver and left kidney tissues were collected and stored in neutral buffered formalin (10%). Right kidney tissue was collected and frozen at -10ºC.

Necropsy of Animals Undergoing Perfusion Fixation
Half of the animals scheduled to be sacrificed at the end of each observation period (10 males and 10 females per treatment group planned) were euthanized by perfusion fixation and underwent a necropsy under the supervision of a Board Certified Veterinary Pathologist.

At the rest of the sacrifice dates (postnatal Days 64, 120 and 364), the animals assigned to perfusion fixation were littermates of the animals assigned for perfusion fixation from the Day 23 cohort.

Histology (Tissues Undergoing Perfusion Fixation)
The following tissues (collected into 10% neutral buffered formalin)
- brain regions (5 locations - cerebrum at the optic chiasm, cerebrum at the base of the posterior hypothalamus, mid-cerebellum and medulla oblongata, pons at the “middle of its protrusion”, and the cranial cervical cord);
- spinal Cord (cervical and thoracic over at least 3 vertebrae each (at two levels));
- lumbar spinal roots (cauda equina);
- dorsal root ganglia;
- sciatic nerve (one proximal and one distal section; one transverse and one longitudinal section at each level); and
- skeletal muscle (gastrocnemius-soleus muscle)
were examined for cellular alterations and other changes, with a particular “emphasis on structural changes indicative of developmental insult”.

Slides were also examined for more typical cellular alterations such as neuronal vacuolation, degeneration necrosis) \and more typical tissue changes such as (astrocytic proliferation, leukocytic infiltration and cystic formation).

Slides were prepared according to GLP consistent with a SOP and the study protocol. Wet tissue was processed, embedded in glycol methacrylate (GMA), sectioned and stained with haematoxylin and eosin (H&E). Tissues were sectioned according to Registry of Industrial Toxicology Animal data guidelines. In appendix I of the final report it is stated that quality checks of the tissue processing were conducted to ensure that it had been appropriate. All slides were then sent for examination by the study veterinary pathologist who was blind to the treatment group.
Other examinations:: Developmental toxicity
Developmental landmarks (i.e., day of vaginal opening for females and day of preputial separation for males) were studied starting on PND 26 in female pups and starting on PND 35 in male pups.
Statistics:: See "any other information on materials and methods incl. tables"
Details on results:: DAMS

Mortality
No 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 weight
The 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 Length
There were no statistically significant differences in gestational length between the different treatment groups.

Clinical Observations
All 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 Consumption
The 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/Period Gestation Lactation
Control 23.0 to 31.5 (67 to 79) 35.1 to 60.6 (99 to 179)
Low Dose 35.9 to 43.7 (103 to 108) 40.1 to 60.9 (114 to 177)
Mid-Dose 42.0 to 45.2 (112 to 123) 40.9 to 69.0 (136 to 201)
High-Dose 27.4 to 31.3 (78 to 80) 39.7 to 70.2 (120 to 211)
Na-citrate 26.2 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 dosage
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.
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.

FOB
During 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.

OFFSPRING

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 Week1 Week7 Week14 Week28 Week49
Low Dose 134% 57% 37% 20% 17%
Mid-Dose 174% 84% 51% 28% 23%
High-Dose 165% 117% - - -

Females
Group Week1 Week7 Week14 Week28 Week49
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
Group/Cohort 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
Group/Cohort 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.
Dose descriptor:: NOAEL
Remarks:: dams
Effect level:: 3 225 mg/kg bw/day (nominal)
Based on:: test mat.
Remarks:: Aluminium citrate (equivalent to 300 mg Al/kg bw/day)
Sex:: female
Basis for effect level:: other: overall effects
Dose descriptor:: NOAEL
Remarks:: adult offspring
Effect level:: 322.5 mg/kg bw/day (nominal)
Based on:: test mat.
Remarks:: Aluminium citrate (equivalent to 30 mg Al/kg bw/day)
Sex:: male/female
Basis for effect level:: other: neuromuscular effect in adults unclear if secondary to body weight or clinical effects
Dose descriptor:: LOAEL
Remarks:: adult offspring
Effect level:: 1 075 mg/kg bw/day (nominal)
Based on:: test mat.
Remarks:: Aluminium citrate (equivalent to 100 mg Al/kg bw/day)
Sex:: male/female
Basis for effect level:: other: neuromuscular effects, hindlimb grip strength, forelimb grip strength in adults unclear if secondary to body weight or other clinical effects, kidney damage through precipitation
Critical effects observed:: not specified
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 damsad libitumviadrinking 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 (), 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 () 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 thetests 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.

FOBobservations 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 theendpoint 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 (30 mg Al/kg bw/day, 100 mg Al/kg bw/day and 300 mg Al/kg bw/day).Asthe 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 of1075 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).

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Quality of whole database:: The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the fact that following absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining, factor the systemic effects of aluminium, including acute toxicity common (refer to endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in the Annex VIII-IX, in accordance with Annex XI, 1.5 of Regulation (EC) No. 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

29 Jun - 26 Sept 1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted before the introduction of GLP and not according to an OECD guideline. However, the study has a proper design and the report is concise.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Pre-guideline study

GLP compliance:

Remarks:

pre-GLP study

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

(exposure room of 10 m³)

Vehicle:

other: unchanged (no vehicle)

Analytical verification of doses or concentrations:

Duration of treatment / exposure:

65 seconds (1x10 and 11x5 seconds) in 6 hours on exposure days

Frequency of treatment:

65 times in 90 days

Remarks:

Doses / Concentrations:
9.0% Aluminiumhydroxichlorid
Basis:
nominal conc.

No. of animals per sex per dose:

Control animals:

yes, concurrent no treatment

Dose descriptor:

LOAEC

Effect level:

15 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Moderate phagocytosis in the lungs and small dust spread into lymph peribronchial lymph nodes

Critical effects observed:

not specified

The daily amount of deodorant sprayed averaged 121.6 g. In the course of 90 days, in total 7785 g of product was sprayed .The entire Locron P consumption is therefore about 700 g. The concentration of deodorant in the exposure room wasapproximately 170 mg/m3 in the first 10 seconds of the first exposure. It is unclear what the average exposure concentration of test substance was.

Study results

Mortality/viability: One male rat died after 62 days of inhalation without clear symptoms

No adverse effects were observed on body weight, blood and urine, as well as albumine/globuline in blood and enzymeactivities. Organ weights were also normal.

Macro and microscopic examination: All animals in the test group showed moderate phagocytose in the lungs and small

dust spread into lymph peribronchial lymph nodes.

Conclusions:

LOAEC = 15.3 mg/m3. Some pathological effects were found: All animals in the test group showed moderate phagocytose in the lungs and small dust spread into lymph peribronchial lymph nodes at the tested concentration and under the conditions of this study.

Executive summary:

The effects of 90-day exposure of rats to deodorant spray containing 9% aluminiumhydroxychloride was tested in this inhalation study. 10 male and 10 female rats were exposed, while another 10 males and females formed the control group.

Clinical signs, body weights, blood and urine and macro and microscopic abnormalities were recorded.

One male rat died after 62 days of inhalation without clear symptoms. No adverse effects were observed on body weight, blood and urine, as well as albumine/globuline in blood and enzymeactivities. Organ weights were also normal.

Macro and microscopic examination showed moderate phagocytose in the lungs and small dust spread into lymph peribronchial lymph nodes in all animals.

A LOAEC of 15.3 mg/m3 could be established (only concentration tested).

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Quality of whole database:: The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the fact that following absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining, factor the systemic effects of aluminium, including acute toxicity common (refer to endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in the Annex VIII-IX, in accordance with Annex XI, 1.5 of Regulation (EC) No. 1907/2006.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

As no studies investigating repeated dose toxicity of reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate are available in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from supporting substances (structural analogues) e.g. aluminium compounds was considered. Aluminium oxide, aluminium hydroxide and aluminium metal are insoluble in water under standard conditions. Based on these physico-chemical characteristics, it is likely that under physiological conditions, the absorption and associated bioavailability of aluminium hydroxide, aluminium oxide and aluminium metal will be low. Following oral absorption, aluminium is present in the body as the ionic species (Al³⁺), which is the determining factor the systemic effects of aluminium, including acute toxicity. Hence, it can be assumed that Al³⁺is the substance of biological interest and the toxicological effects can be attributed mainly to Al³⁺.

Following absorption of the substance used for read-across like aluminium salts (e.g., aluminium nitrate, aluminium chloride, aluminium sulphate, etc.) aluminium is present in the body as Al³⁺as well. Therefore, with appropriate consideration of bioavailability differences, it is reasonable to consider data obtained from aluminium salts, generally more soluble, in the hazard identification of the highly soluble aluminiumsulfatenitrate.

In conclusion, in terms of hazard assessment of toxic effects, available data for human health endpoints for various aluminium compounds can be read-across to reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al³⁺) (Krewski et al., 2007; ATSDR, 2008).

A detailed justification read-across is provided in the technical dossier (see IUCLID Section 13) as well as in the Chemical Safety Report (see Part B).

Repeated dose toxicity: oral

Since no studies investigating the repeated dose toxicity (oral route) of reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate are available in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from supporting substance (structural analogue), aluminium citrate (31142-56-0) was performed. Read-across is justified based on the fact that following oral absorption, aluminium is present in the body as the ionic species (Al³⁺), which is the determining factor the systemic effects of aluminium, including repeated dose toxicity oral.

CAS 31142-56-0 (aluminium citrate)

The most relevant and reliable key study in the developmental neurotoxicity and chronic toxicity study was performed by ToxTest. Alberta Research Council Inc. (2009), groups of Sprague-Dawley rats were exposed to 322.5, 1075 and 3225 mg/kg bw/day of aluminium citrate (equivalent to 30, 100 and 300 mg Al/kg bw/day) via drinking water. Pregnant animals were exposed during gestation (starting on gestational day 6) through weaning, and the offspring was exposed during post-weaning and continuing up to post-natal day 364.

No relevant signs of systemic toxicity (mortality, clinical signs, body weight, water consumption, Functional Observational Battery (FOB)) were observed in the dams exposed up to the highest dose. Therefore, 3225 mg/kg bw/day of aluminium citrate (equivalent to 300 mg Al/kg bw/day) was considered the systemic NOAEL for dams in this study. However, no necropsy and histopathological examinations were performed in these animals.

In offspring animals exposed up to post-natal day 364, a significant decrease in post-weaning body weight was observed in the high dose aluminium citrate group in both males and females. Urinary tract pathology was observed in high and to a certain extent also in mid dose rats, more frequently in the males. The results showed no evidence of an effect on memory, motor activity, learning, or any other neurobehavioural effects, with one exception, that the study investigated in detail. Also extended neuropathological examination did not show any treatment related effects. The only effect related to neuromuscular function that was observed in this study was a reduced fore- and hindlimb grip strength in both sexes of the mid and high dose group and a reduction in foot splay in females of the mid and high dose group in adult offspring (but not in neonatal and juvenile animals). However, according to the authors, the latter findings may also be related to body weight differences and cannot unequivocally be attributed to neurotoxicity. The most prominent findings in this studies were urinary tract lesions at necropsy (4 males, 1 female in the mid dose group, 8 females and 23 males in the high dose group). Other findings in the high dose group included decrease in body weight in animals exposed up to post-natal day 120; defecation (more boluses produced by females compared to the controls); urination (males produced more urine pools that controls); tail pinch (females displayed more exaggerated responses); the albumin/globulin ratio (males exposed up to post-natal day 64 had a greater mean ratio than the controls). A LOAEL of 1075 mg/kg bw/day of aluminium citrate (equivalent to 100 mg Al/kg bw/day) was assigned based on this study. Accordingly, the NOAEL based on kidney and possible neuromuscular effects was 322.5 mg/kg bw/day of aluminium citrate (equivalent to 30 mg Al/kg bw/day).

Additionally, data from structurally related substances were used as supporting information.

CAS 1327-27-2 (aluminium chloride, basic)

In the combined repeated dose and reproduction/developmental screening study by Beekhuijzen (2007), treatment with aluminium chloride basic by oral gavage in male and female Wistar rats at dose levels of 3.6, 18 and 90 mg Al/kg bw/day revealed local toxic effects at the highest dose, comprising inflammation of the glandular stomach mucosa. Based on these findings, the LOAEL for local effects was 90 mg Al/kg bw/day, and the local NOAEL was 18 mg Al/kg bw/day. No systemic effects were observed up to the highest dose. Thus, the systemic NOAEL was higher or equal to 90 mg Al/kg bw/day

CAS 7784-27-2 (aluminium nitrate nonahydrate)

In another study, the oral toxicity of aluminium nitrate nonahydrate was examined in a subchronic 100-days study in Sprague-Dawley female rats. Aluminium nitrate nonahydrate was given orally via drinking water at 0, 360, 720 and 3600 mg/kg bw/d, which corresponds to 0, 26, 52 and 260 mg/kg bw/d when expressed as aluminium dose. Nutritional and toxicological parameters were monitored throughout the study. At the end of the study, animals were sacrificed and assessed for gross pathology and histopathological changes. At 260 mg Al/kg bw/d a statistically significant decrease in body weight gain and food and water consumption was observed. All other toxicological parameters measured were not or not significantly changed in relation to the exposure. At 26 and 52 mg Al/kg bw/d no significant toxic effects were reported, therefore the NOAEL was 52 mg Al/kg bw/d via drinking water (Domingo et al., 1987).

CAS 7784-31-8 (aluminium sulphate octadecahydrate)

The subacute oral toxicity of aluminium sulphate (and potassium aluminium sulphate) was investigated in albino male rats. Aluminium salts were dissolved in deionized water and administered daily for up to 21 days by gavage. The calculated aluminium doses given were 1.72, 2.16, 2.86, 4.29, 8.59, and 17.18 mg/kg bw (potassium aluminium sulphate was administered only at 2.86 and 4.29 mg aluminium/kg bw). Five animals per dose were sacrificed after 7, 14, and 21 days, respectively, and assessed for histopathological changes of selected organs. Dose- and/or time-related effects were observed in liver, kidney, brain, testes, bone and stomach. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 1.72 mg aluminium/kg bw, based on cytotoxic effects in the liver (Roy et al., 1991)

Repeated dose toxicity: inhalation

Since no studies investigating the repeated dose toxicity (inhalation route) of reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate are available in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from supporting substance (structural analogue), aluminium hydroxychloride (CAS 12042-91-0) was performed. Read-across is justified based on the fact that following oral absorption, aluminium is present in the body as the ionic species (Al³⁺), which is the determining factor the systemic effects of aluminium, including repeated dose toxicity inhalation.

CAS 2042-91-0 (aluminium hydroxychloride)

The effects of 90-day exposure (limit test) of rats to deodorant spray containing 9% aluminium hydroxychloride was tested in a inhalation study non GLP compliant and conducted with a method similar to OECD Guideline 413. 10 male and 10 female rats were exposed to the test substance, while another 10 males and females formed the control group.

Clinical signs, body weights, blood and urine and macro and microscopic abnormalities were recorded.

Macro and microscopic examination showed moderate phagocytosis in the lungs and small dust spread into lymph peribronchial lymph nodes in all animals.

A LOAEC of 15.3 mg/m³ could be established (only concentration tested).

Additionally, data from a structurally related substance were used as supporting information

Aluminium powder and aluminium oxide

Gross et al. (1973) exposed rats, guinea pigs and hamsters to three different aluminium powders (British pyro powder, a US-flake powder, and a US-source atomized powder with approximately spherical particles) and also aluminium oxide dust, included as a negative “non-fibrogenic control”. The Al₂O₃content was 16.6% for the British pyro powder, not stated for the flake powder and 2.9% for the atomized powder. The doses administered by inhalation ranged from 15 to 100 mg/m³, 6 hours per day, 5 days per week for either 6 or 12 months. Thirty rats were exposed to pyro powder at each 15, 30, 50 and 100 mg/m³, 30 rats were exposed to atomized metal powder at each 15, 30, 50 and 100 mg/m³, 30 rats were exposed to flake powder at 15 and 30 mg/m³, and 30 rats were exposed to aluminium oxide dust at 30 and 70 mg/m³. Five rats were sacrificed per time point (6, 8, 12 and 18 months). Thirty hamsters were exposed to pyro powder at 50 and 100 mg/m³, 30 hamsters were exposed to atomized powder at 50 and 100 mg/m³, and 30 hamsters were exposed to aluminium oxide at 70 mg/m³. Between 15 and 25 guinea pigs were exposed to each of the aluminium powders at 15 and 30 mg/m³. Twelve guinea pigs were exposed to aluminium oxide dust at 30 mg/m³. The chambers were approximately 1.2 m³ in volume, moisture was removed using anhydrous calcium chloride and powders were dispersed through the chambers by means of a dust-feed mechanism (Wright). Air flow was limited to10 litres/min to attain high dust concentrations.

The dusts, suspended in tap water, were also administered by intratracheal instillation to different groups of animals. Concentrations were used such that 1 mL of the suspension contained the required dose. Injections were performed under anaesthetic (ether) using an illuminated laryngeal speculum to facilitate the introduction of the 18-gauge, blunt needle. A tap water “vehicle” control group was included. For intratracheal instillation, 15 rats and 15 hamsters were allocated to each dose for the pyro, atomized and flaked powders. With the exception of the highest dose level, 1 to 5 animals were sacrificed at 6 months and 7 to 10 animals at 12 months post-exposure. At the 100 mg/m³ dose level for the pyro powder, 15 animals were dosed, 4 were sacrificed at 2 months, 4 at 4 months and 7 at 6 months. At the 100 mg/m³ dose level for the atomized powder, 15 animals were dosed, 3 animals were sacrificed at 2 months, 3 animals at 4 months and 2 animals at 6 months.

Mortality was reported but no data on clinical signs, body weight, or organ weights was provided. Histopathological examinations of the lungs were conducted on sections cut in triplicate from lung tissue stained with either eosin alone to show aluminium particles, hematoxylin-eosin, or PAS/ van Gieson. To show cellular components and stromal support structures, the haematoxylin-eosin stained sections were examined before and after decolorization and impregnation with silver (Gordon and Sweets method).

Intratracheal injection of the aluminium powders caused nodular pulmonary fibrosis in the lungs of the rats only at the highest dose administered (100 mg). A fibrotic response was not observed in hamsters indicating inter-species differences in response. 12 mg of dust administered intratracheally did not lead to collagen production in rats or hamsters. The response of hamster and guinea pigs lungs differed from rats. At higher concentrations, hamster and guinea pig lungs developed metaplastic foci of alveolar epithelium that persisted beyond the resolution of alveolar proteinosis and clearance of the dust particles.

Progressive fibrosis was not observed in rats on inhalation exposure to the powders indicating that the intratracheal instillation mode of test compound delivery may lead to artifacts not representative of physiologically relevant exposures. There was no dose response evident or a noticeable difference between responses to the different aluminium powders. All three species developed widespread alveolar proteinosis, rats exhibiting the most severe response. However, alveolar walls appeared thin and normal. The proteinosis resolved progressively after cessation of exposure. Small scattered foci of endogenous lipid pneumonitis (granulomatous inflammation) developed associated with cholesterol crystals that were not surrounded by alveolar proteinaceous material. These effects generally occurred in regions not associated with dust particles and left small collagenous scars. The group of rats exposed for 12 months to 15 mg/m³ of aluminium powder showed moderate alveolar proteinosis after 6 months of exposure. Granulomatous inflammation was observed at 50 mg/m³ after about 3 months of exposure.

Overall, there was no consistent relationship between dose and severity of response for any of the aluminium powders. The results showed no clear difference in reaction to the different powders. The results from this study do not provide evidence to support a progressive fibrotic response on inhalation exposure to aluminium powder. No alveolar proteinosis or thickening of alveolar walls was observed in rats, hamsters or guinea pigs exposed to Al₂O₃dust (66% < 1 μm) included in the study as a “non-fibrogenic” control.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Hazard assessment is conducted by means of read across from a structural analogue aluminium citrate (31142-56-0). The available study is adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Hazard assessment is conducted by means of read across from a structural analogue aluminium hydroxychloride (12042-91-0). The available study is adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details)

Justification for classification or non-classification

The available data on repeated dose toxicity of structurally related substances to reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and therefore are conclusive but not sufficient for classification.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Parameter	Pairwise Differences
Absolute Agranulocytes	Ns
Absolute Granulocytes	The high dose group was significantly greater than the controls and low dose group (p=0.0240 and p=0.0354, respectively)
Agranulocytes	Significant group effect but no pair-wise comparisons with p-values<0.05.
Granulocytes	Significant group effect but no pair-wise comparisons with p-values<0.05.
HCT (haematocrit)	The high dose group was significantly lower than the controls and the low dose group (p=0.0113 and p=0.0238, respectively). The Na-citrate group was significantly lower than the control group (p=0.0365).
HGB (haemoglobin)	The high dose group was significantly lower than the control and the low dose group (p=0.0181 and p=0.0202, respectively).
MCH (mean cell haemoglobin)	The high dose group was significantly lower than all the other groups (controls, p<0.0001; low dose group, p=0.0009; mid-dose group, p=0.0005; Na-citrate group, p=0.0010).
MCHC (mean cell haemoglobin concentration)	Ns
MCV (mean cell volume)	The high dose group was significantly lower than all the other groups (controls, p<0.0001; low dose group, p=0.0007; mid-dose group, p=0.0005; Na-citrate group, p=0.0012).
PLT (platelet count)	Ns
NUC_RBC (nucleated red blood cells)	Zero
RBC (red blood cell count)	The high dose group was significantly greater than the mid-dose group (p=0.0341) and the Na-citrate group (p=0.0034).
WBC (white blood cell count)	Ns

Parameter	Units	Day 23	Day 64	Day 120	Day 364
ALB (albumin)	g/L	34.5 (1.51)	45.00 (1.89)	50.27 (2.33)	48.25 (3.62)
ALP (alkaline phospha-tase)	U/L	330.30 (36.32)	119.20 (21.40)	52.91 (19.03)	36.25 (18.12)
ALT (alanine aminotrans-ferase)	U/L	28.80 (4.32)	23.70 (5.46)	20.45 (4.55)	25.00 (3.55)
AST (aspartate aminotrans-ferase)	U/L	173.10 (48.21)	81.00 (17.40)	74.55 (9.68)	108.88 (44.96)
A_G (albumin/ globulin ratio)		2.55 (0.33)	2.68 (0.31)	2.52 (0.16)	1.95 (0.30)
CA (calcium)	mM	2.86 (0.05)	2.76 (0.08)	2.71 (0.08)	2.67 (0.10)
CHOL (cholesterol)	mM	2.60 (0.39)	2.09 (0.49)	1.85 (0.38)	3.68 (0.86)
CK (creatinine kinase)	U/L	972.20 (479.79)	414.30 (109.88)	308.55 (132.96)	438.25 (336.60)
CL (chloride)	mM	101.40 (2.17)	99.60 (2.80)	102.64 (1.03)	100.00 (1.41)
CRE (creatinine)	µM	12.70 (5.40)	29.20 (3.97)	42.27 (6.68)	41.13 (4.97)
GLOB (globulin)	g/L	13.70 (1.64)	17.00 (2.00)	20.00 (1.41)	25.00 (2.33)
GLU (glucose)	mM	10.18 (1.27)	12.80 (1.68)	11.15 (1.11)	9.25 (2.09)
K (potassium)	mM	5.11 (0.24)	4.22 (0.38)	4.55 (0.42)	4.30 (0.44)
Na (sodium)	mM	137.40 (1.71)	141.00 (2.45)	141.55 (2.21)	144.88 (2.70)
Phos (phosphorus)	mM	2.66 (0.22)	2.23 (0.39)	1.92 (0.25)	1.73 (0.35)
SDH (Sorbitol dehydrog-enase)	U/L	52.10 (9.50)	35.30 (7.20)	36.09 (17.54)	66.25 (21.53)
TBIL (total bilirubin)	µM	1.50 (0.53)	2.00 (0.47)	2.73 (0.47)	2.75 (0.46 )
TG (triglycerides)	mM	1.62 (0.53)	1.85 (0.82)	3.91 (3.42)	6.16 (6.52)
TP (total protein)	g/L	48.20 (2.04)	62.00 (3.23)	70.27 (3.23)	73.25 (3.11)
Urea	mM	5.99 (1.20)	6.14 (1.26)	4.95 (0.58)	5.38 (1.08)

Parameter	Units	Day 23	Day 64	Day 120	Day 364
ALB (albumin)	g/L	34.40 (1.65)	37.60 (1.90)	38.67 (3.24)	36.00 (4.90)
ALP (alkaline phospha-tase)	U/L	332.50 (51.70)	203.30 (33.45)	87.78 (15.78)	70.00 (16.84)
ALT (alanine aminotrans-ferase)	U/L	26.80 (4.54)	29.50 (7.85)	29.56 (12.64)	57.00 (39.55)
AST (aspartate aminotrans-ferase)	U/L	151.70 (12.98)	105.00 (21.85)	83.78 (16.32)	134.75 (53.51)
A_G (albumin/ globulin ratio)		2.49 (0.21)	1.97 (0.21)	1.58 (0.14)	1.23 (0.21)
CA (calcium)	mM	2.85 (0.08)	2.72 (0.10)	2.65 (0.04)	2.66 (0.14)
CHOL (cholesterol)	mM	2.47 (0.31)	1.92 (0.41)	2.03 (0.33)	3.70 (1.32)
CK (creatinine kinase)	U/L	806.10 (190.93)	633.40 (149.19)	387.33 (152.60)	557.50 (174.88)
CL (chloride)	mM	99.70 (2.00)	99.10 (1.66)	102.33 (1.12)	101.25 (1.39)
CRE (creatinine)	µM	10.60 (3.81)	19.90 (4.09)	30.11 (5.46)	40.63 (9.10)
GLOB (globulin)	g/L	13.90 (1.10)	19.30 (2.11)	24.56 (0.73)	29.50 (2.83)
GLU (glucose)	mM	8.88 (1.22)	12.49 (2.24)	12.74 (1.82)	9.60 (1.22)
K (potassium)	mM	5.00 (0.35)	4.57 (0.30)	4.49 (0.29)	4.94 (0.49)
Na (sodium)	mM	136.80 (1.75)	141.70 (1.42)	142.33 (1.00)	146.00 (3.89)
Phos (phosphorus)	mM	2.51 (0.19)	2.60 (0.26)	2.05 (0.13)	2.03 (0.47)
SDH (Sorbitol dehydrog-enase)	U/L	51.90 (8.70)	52.70 (17.95)	33.44 (15.80)	72.50 (39.58)
TBIL (total bilirubin)	µM	1.30 (0.48)	1.70 (0.48)	2.56 (0.53)	3.13 (1.46)
TG (triglycerides)	mM	2.10 (1.23)	2.20 (0.51)	3.13 (1.07)	2.96 (1.41)
TP (total protein)	g/L	48.30 (2.11)	56.90 (3.14)	63.22 (3.35)	65.50 (5.48)
Urea	mM	5.23 (1.23)	7.07 (1.26)	4.88 (0.70)	5.74 (1.34)

Parameter	Day 23	Day 64	Day 120	Day 364
ALB (albumin)	Log transform-ation required.	High < control (p=0.0002), low (p=0.0014) and mid dose (p=0.0005).	High dose < low (p=0.0087) and mid dose (p=0.0028) groups.
ALP (alkaline phospha-tase)		High> control, low-dose and mid-dose (p<0.0001) High dose>Na-citrate (p<0.0001)	High dose > control (p=0.0013), low dose (p=0.0071), and mid-dose (p=0.0300)
ALT (alanine aminotrans-ferase)
AST (aspartate aminotrans-ferase)	Log transformed.
A_G (albumin/ globulin ratio)
CA (calcium)	High > control (p=0.0117). Na-citrate group < mid dose (p=0.0038) and high dose groups (p=0.0001).	High> control, low-dose and mid-dose (p<0.0001) High dose>Na-citrate (p<0.0001)	High > control (p=0.0201). High > Na-citrate (p=0.0045)
CHOL (cholesterol)
CK (creatinine kinase)
CL (chloride)			Na-citrate < control (p=0.0051)	Na-citrate < control (p=0.0038) and low dose (p=0.0256)
CRE (creatinine)		All adjusted p values >0.05.	All adjusted p-values <0.05)
GLOB (globulin)		High < control (p=0.0026), low-dose (p=0.0189) and mid-dose (p=0.0004). High dose<Na-citrate (p=0.0484)	High < mid dose (p=0.0339)
GLU (glucose)	Control > high dose group (p=0.0214) & low dose group (p=0.0447). Na-citrate < control (p=0.0007)	All adjusted p-values > 0.05
K (potassium)	Control>low dose group (p=0.0463). Na-citrate <control (p=0.0018)			All adjusted p-values >0.05.
Na (sodium)	Na-citrate group > control (p<0.0001), low dose (p<0.0001), mid-dose (p<0.0001) and high dose (p=0.0069).	Mid > high dose (p=0.0103) Mid-dose > Na-citrate (p=0.0168).
Phos (phosphorus)	Control > high dose group (p=0.0009)
SDH (Sorbitol dehydrog-enase)
TBIL (total bilirubin)	Categorical.
TG (triglycerides)		High dose < control (p=0.0047) and low dose (p=0.0145).
TP (total protein)	Log transformed.	High < control (p=0.0001), low (p=0.0012) and mid-dose groups (p<0.0001). High dose < Na-citrate (p=0.0371).	High <control (p=0.0330), low (p=0.0061) and mid-dose (p-=0.0013)
Urea	Log transformed. Na-citrate > mid- (p=0.0208) and high dose (p=0.0405) groups.		High dose > control (p=0.0173) and low dose (p=0.0366).	High dose > control (p=0.0154), low dose (p=0.0261), and mid-dose (p=0.0067).

Parameter	Day 23	Day 64	Day 120	Day 364
ALB (albumin)
ALP (alkaline phospha-tase)	High dose > control (p=0.0268)	High dose > control (p=0.0002), low (p=0.0002) and mid (p=0.0184) dose groups. High dose > Na-citrate group (p<0.0001)
ALT (alanine aminotrans-ferase)
AST (aspartate aminotrans-ferase)	Na-citrate>low dose (p=0.0048)
A_G (albumin/ globulin ratio)		High dose > control, low and mid dose groups (p<0.0001) Mid dose > control (p=0.046). High dose > Na-citrate group (p<0.0001)	Na-citrate > control (p=0.0303)
CA (calcium)		High dose > control, low and mid dose groups (p<0.0001) High dose > Na-citrate group (p<0.0001)
CHOL (cholesterol)
CK (creatinine kinase)
CL (chloride)	All adjusted p-values >0.05.	High dose < control (p=0.0003), low (p<0.0001)and mid dose (p=0.0012) groups . High dose < Na-citrate (p=0.0073)
CRE (creatinine)		High dose > control, low and mid dose groups p<0.0001). High dose > Na-citrate (p<0.0001)
GLOB (globulin)		High dose < control (p<0.0001), low (p<0.0001) and mid dose (p=0.0002) groups. High dose < Na-citrate (p=0.0008)	Na citrate < control (p=0.0003), low dose (p=0.0076), and mid-dose (p=0.0052).

Group	Sex	Al µg/g, mean (sd)
Control	F	0.26 (0.24)
Low dose	F	0.19 (0.06)
Mid-dose	F	0.41 (0.22)
High dose	F	3.43 (0.21)
Na-citrate	F	0.13 (0.04)
Control	M	0.23 (0.15)
Low dose	M	0.19 (0.08)
Mid-dose	M	0.54 (0.24)
High dose	M	6.72 (4.78)
Na-citrate	M	0.14 (0.03)