Registration Dossier

Toxicological information

Endpoint summary

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Description of key information

chronic toxicity:

oral, rat: NOAEL: 322.5 mg/kg bw/day as aluminium citrate (equivalent to 113.36 mg Al oxide/kg bw/day)

inhalation, rat: NOAEC >= 75 mg/m³ as aluminium oxide

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:
experimental study
Adequacy of study:
weight of evidence
Study period:
2008-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD TG 426 and OECD TG 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. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 bythe 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 (e.g. ad libitum): 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 (e.g. ad libitum): Deionised H2O (or the dosing solutions) was provided 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°C
- Humidity (%): 30-70%;
- Air changes (per hr): ≥ 10 per hour in the room
- Photoperiod (hrs dark / hrs light): 12 hr. light
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:
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.

Dams
GD 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 1-64
Cohort 3 – GD 6-21, PND 1-120
Cohort 4 – GD 6-21, PND 1-364


Frequency of treatment:
Ad libitum (Daily, 7 days per week)
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Al; corresponding to 322.5 mg /Al citrate/kg bw/day (Group A)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Al; corresponding to 1075 mg /Al citrate/kg bw/day (Group D)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Al; corresponding to 3225 mg/Al citrate/ kg bw/day (Group E)
No. of animals per sex per dose:
Dams: 20/group;
Offspring: 80 females and 80 males/group;
Litters: 20 litters/group.
Control animals:
yes, concurrent no treatment
other: A group of animals exposed to Na citrate at a citrate concentration equimolar to the high dose aluminium group was included to investigate the possibility of counter ion effects.
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 mothods incl. tables"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see "Details on results"
Mortality:
mortality observed, treatment-related
Description (incidence):
pups only; see "Details on results"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
pups only; see "Details on results"
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
see "Details on results"
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
see "Details on results"
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
pups only; see "Details on results"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
pups only; see "Details on results"
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
pups only; see "Details on results"
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
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:
systemic toxicity
Effect level:
322.5 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
corresponding to 30 mg Al/kg bw/day
Sex:
male/female
Basis for effect level:
other: neuromuscular effects (hindlimb grip strength, footsplay)
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
1 075 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
corresponding to 100 mg Al/kg bw/day
Sex:
male/female
Basis for effect level:
other: neuromuscular effects (hindlimb grip strength, footsplay)
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 3 225 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
corresponding to 300 mg Al/kg bw/day
Sex:
female
Basis for effect level:
other: no adverse effects observed at highest dose tested
Critical effects observed:
not specified

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

Day 23 cohort, females: The low dose group had significantly lower mean cell volume (MCV) than the control group (p=0.0189). The platelet count (PLT) was significantly lower in the low dose group than in the high dose group (p=0.0418). Nucleated red blood cells (NUC-RBC) in the low dose group differed significantly from this parameter in the control, mid-dose and high dose groups (p=0.0363, p=0.0101, and p=0.0062, respectively).

 

Day 23 cohort, males: The high dose group had marginally higher MCV than the control group (p=0.050).

 

Day 64 cohort, females:

 

Day 64 cohort, males:

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

 

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.

 

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

 

Overall, effects in the Day 23 cohort were not considered clinically significant. In the Day 64 cohort, however, both males and females in the high dose group showed low grade microcytic anaemia. The anaemia had resolved in the females by cohort Day 364.

 

Coagulation parameters:

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

Clinical Chemistry

Clinical Chemistry – Serum Parameter Values in the control groups (10 animals/group)

 

Female Controls (mean (standard deviation))

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)

 

Male Controls (mean (standard deviation))

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)

 

 

Statistically significant differences from the pair-wise comparisons are provided in the table below. Pair-wise comparisons were only conducted where a significant effect of group was found in the ANOVA. Results from comparisons between the control and the different aluminium citrate groups are in bold font.

 

FEMALES

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).

 

Statistically significant differences from the pair-wise comparisons in the male animals are provided in the table below. Pair-wise comparisons were only conducted where a significant effect of group was found in the ANOVA. Results from comparisons between the control and the different aluminium citrate groups are in bold font.

MALES

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).

 

GLU (glucose)

 

High dose < control (p=0.0207), low (p=0.0029) and mid dose (p=0.0136)groups

 

 

K (potassium)

 

 

 

 

Na (sodium)

Na-citrate> control (p<0.0001), low dose (p=0.0006), mid-dose (p=0.0005), and high-dose (p=0.0421) groups.

High dose < control (p=0.0247), low dose (p=0.0008), and mid-dose (p=0.0118) groups.

 

 

Phos (phosphorus)

 

High dose > control (p=0.0003), low (p=0.0097) and mid dose (p=0.0046)groups.

 

High dose > Na-citrate (p=0.0031)

 

 

SDH (Sorbitol dehydrog-enase)

 

 

 

 

TBIL (total bilirubin)

 

Na-citrate <low dose, mid-dose and high dose.

 

 

TG (triglycerides)

 

High dose < control (p=0.0208)and low dose (p=0.0023)

 

 

TP (total protein)

 

High dose < control (p=0.0002), low dose (p=0.0008), and mid dose (p=0.0105)

Na-citrate < control (p=0.0096) and low dose (p=0.0276)

 

Urea

 

High dose > control (p=0.0001), low dose (p<0.0001), and mid dose (p=0.0003)

 

Na-citrate < high dose (p<0.0001)

 

 

 

In summary, significant elevations were observed predominantly in ing the high dose group relative to the other groups. Serum chemistry changes associated with aluminum toxicity such as elevated alkaline phosphatase and serum calcium were observed. The authors state the levels still remained within the normal range. Effects were most pronounced in the Day 64 cohort animals.

Tissue Metal Levels

Neonatal Pups (PND 4)

Group

Sex

Alµg/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)

 Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control group. This provides evidence for vertical transmission of Al to pups in-utero. There were no significant sex differences.

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 viadrinking 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 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). 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
113 mg/kg bw/day
Study duration:
chronic
Species:
rat
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 presence of a common metal ion, or ion complex including a hydrated metal ion, and following from this a similar chemical behaviour (refer to endpoint discussion for further details).
The available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
System:
nervous system
Organ:
not specified

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
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:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
: Only one sex of animals; Number of animals per group (sex/dose/timepoint); Outcomes assessed (lack of observations of body weight and other clinical signs); lack of information on animal husbandry
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Details on test animals and environmental conditions: no data
It is unclear whether the animals remained in the inhalation chambers even when the exposure was not occurring.

Diet and water: NR

Acclimation and monitoring animal health:
No information was provided on acclimation or animal care.

Route of administration:
other: Inhalation: dust and Intratracheal injections
Type of inhalation exposure:
whole body
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: No information was provided on the MMAD and GSD.

Further detail on particle characteristics (e.g. shape):
Particle size by count was provided for the size ranges < 1.0 µm; 1 to 4 µm; and > 4 µm.
(1) British pyro powder
Shape: flake-like
Size: < 1 µm 4.2%, 1 – 4 µm 87.3%, > 4 µm 8.5%
Mean diameter: 2.49 µm
Specific surface area: 10.4 m²/g

(2) US –source atomised particles.
Shape: “spherical”
Size: < 1 µm 1.5%, 1 – 4 µm 95.6%, > 4 µm 2.9%;
Mean diameter: 2.22 µm
Specific surface area: 0.8 m²/g

(3) US-source flake powder
Shape: flake
Size: < 1 µm 0.0%, 1 – 4 µm 28.6%, > 4 µm 71.4%;
Mean diameter: 4.85 µm
Specific surface area: 8.4 m²/g

(4) Negative control: aluminium oxide dust.
Shape: not stated
Size: < 1 µm 66%, 1 – 4 µm 25%, > 4 µm 9%;
Mean diameter: 0.80 µm
Specific surface area: 6.3 m²/g
Details on inhalation exposure:
Further details on inhalation exposure:
The chambers were approximately 1.2 m³ in volume. Moisture was removed using anhydrous calcium chloride. Powders were dispersed through the chambers by means of a dust-feed mechanism (Wright). Air flow was limited to 10 litres/min to attain high dust concentrations.

Details on Intratracheal Instillation:
A suspension of the dust in tap water was instilled intratracheally. 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.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The air flow through the chambers was monitored with outflowing chamber air forced through 20.3 x 25.4 cm filter (Millipore) filters before ventilation. The filters were used to gravimetrically estimate the average dust concentration in the chamber each day. The data were not reported however.
Duration of treatment / exposure:
Inhalation
Rats in the 50 and 100 mg/m³ chambers were exposed for 6 months. Exposure duration was 12 months for the animals at the lower aluminium powder concentrations of 15 and 30 mg/m³.

The aluminium oxide control rats were exposed to 75 mg/m³ for six months. An additional 30 rats and 12 guinea pigs were exposed to 30 mg/m³ of aluminium oxide dust for a year.
Frequency of treatment:
Inhalation
6 hr/day; 5 days a week
Dose / conc.:
15 mg/m³ air
Remarks:
pyro powder, atomized powder, flake powder
Dose / conc.:
30 mg/m³ air
Remarks:
pyro powder, atomized powder, flake powder
Dose / conc.:
50 mg/m³ air
Remarks:
pyro powder, atomized powder
Dose / conc.:
100 mg/m³ air
Remarks:
pyro powder, atomized powder
Dose / conc.:
30 mg/m³ air
Remarks:
aluminium oxide
Dose / conc.:
75 mg/m³ air
Remarks:
aluminium oxide
No. of animals per sex per dose:
Inhalation
Rats:
30 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
30 rats were exposed to aluminium oxide dust at 30 and 75 mg/m³

- 5 animals were sacrificed per time point (6, 8, 12 and 18 months).

Intratracheal instillation **
15 rats 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.

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 were sacrificed at 2 months, 3 at 4 months and 2 at 6 months.
Control animals:
yes
Details on study design:
Control animals:
50 rats were untreated (laboratory controls). Five animals were examined per time point.
Aluminium oxide (numbers and dosing described above) were included as “non-fibrogenic” controls.
For the intratracheal instillation group, 15 rats were included as vehicle controls.

No information was provided on the method used to allocate the animals to groups.
Positive control:
No.
Observations and examinations performed and frequency:
Observations and examinations performed:
No information was provided on observations to monitor animal health but mortality was recorded.

Frequency of the observations and examinations:
For the inhalation exposure, pathological examinations took place at 6, 8, 12 and 18 months into the experiment for the 50 and 100 mg/m³ aluminium powder dose groups and the 70 mg/m³ aluminium oxide dose group (i.e. 0, 2, 6 and 12 months after cessation of exposure). Kills of the lower dose animals took place at 6 and 12 months (0 and 6 months post-exposure).
For the intratracheal instillation, see ** above.
Sacrifice and pathology:
The method used to sacrifice the animals was not reported in the article.
Histopathological examinations of lung tissue were conducted using sections cut in triplicate and embedded in paraffin blocks. One section was stained with eosin to show aluminium particles, a second section was stained with hematoxylin-eosin, and a third section with PAS or van Gieson. To show cellular components and stromal support structures, the hematoxylin-eosin stained sections were photographed then decolorized and impregnated with silver (Gordon and Sweets method) before another photograph was taken. Aluminium particles were removed prior to this procedure using 10% sodium bisulfite.
Other examinations:
No data.
Statistics:
No information was provided on statistical methods used for comparing mortality rates.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
A higher rate than ideal was observed. As high rates were observed in control groups as well as treated groups and appeared to show no relationship to dose, deaths were unlikely to be simply related to the dust exposure. The authors suggest an effect of crowding and low air flow in the chambers. Quantitatively, the mortality results from this study are not reliable. The absence of observations of clinical signs also suggests that caution ought to be used in interpreting the results.

ORGAN WEIGHTS
Lung weights were measured but not reported.

HISTOPATHOLOGY: NON-NEOPLASTIC
ADEQUATE
- alveolar proteinosis was observed after 6 months exposure to 15 mg/m³ in rats.
- foci of fibrosis found for pyro Al powder at 50 mg/m³ exposed for 108 days; killed 6 months later.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Pulmonary lymphoid tumors, reticulum cell and lymphosarcoma were noted in both the experimental and control groups. These were interpreted as spontaneous tumors in aging rats not associated with pulmonary dust exposure.




Dose descriptor:
LOAEC
Remarks:
local effects
Effect level:
15 mg/m³ air
Based on:
test mat.
Remarks:
Al powder
Sex:
not specified
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
>= 75 mg/m³ air
Based on:
test mat.
Remarks:
Al2O3 dust
Sex:
not specified
Basis for effect level:
other: no adverse effect observed at the highest dose tested
Critical effects observed:
not specified

Inhalation series:

Mortality: 

Spontaneous deaths were more numerous among all 3 species than ideal. The % of the animals dead at 6 months and 12 months are provided in the table below. The numbers are extracted from Tables 3, 4 and 5 of the publication.

 

Animal

Dust Type

Dose (mg/m³)

Exposure duration

% dead: 6 mos.

% dead: 12 mos.

Rats

Atomised Al

100

6 mos

0

0

 

Atomised Al

50

6 mos

7

25

 

Atomised Al

30

12 mos

0

28

 

Atomised Al

15

12 mos

0

8

 

Pyro Al

100

6 mos

0

40

 

Pyro Al

50

6 mos

0

20

 

Pyro Al

30

12 mos

0

20

 

Pyro Al

15

12 mos

3

36

 

Flake Al

30

12 mos

0

24

 

Flake Al

15

12 mos

0

32

 

Al2O3

75

6 mos

0

0

 

Al2O3

30

12 mos

0

20

Air control

0

6 mos

0

0

Air control

0

12 mos

0

0

 

 

Lung histology

Al-powders:

All three species developed alveolar proteinosis (AP);

 

Rats:

50 and 100 mg/m³ exposed for 6 mths:

Marked AP; but alveolar walls were generally thin and appeared normal;

AP underwent spontaneous resolution with little evidence remaining 1.5 years post-exposure.

15 and 30 mg/m³ for 12 mths:

Moderate AP from 6 to 12 months followed by gradual clearing. Some AP still present at 24 mths.

 

Persistent changes:

Small scattered foci of endogenous lipid pneumonitis (granulomatous inflammation) associated with cholesterol crystals that were not surrounded by AP material. These occurred generally not in regions with dust particles. The foci left collagenous scars.

No carcinoma was observed. Lymphoid tumors, reticulum cell and lymphosarcoma noted in both the treated and control groups. Considered spontaneous by authors and numbers were not provided.

 

 

Al2O3:

Rats:

Small foci concentrated in respiratory bronchioles and alveolar ducts – consisting of clustered alveoli with swollen macrophages engorged with particles; no thickening of alveolar walls evident; no evidence of AP or pnuemonitis.

 

Distribution and clearance of dust:

Dust remained finely dispersed even within the cytoplasm of macrophages.

Rats:

50 and 100 mg/m³ exposed for 6 mths: Clearance by 1.5 years post-exposure

15 and 30 mg/m³ exposed for 12 mths: some finely dispersed Al-powder particles were still evident 1 year post-exposure.

 

There was no dose response evident or noticeable differences in response to the different aluminium powders.

 

The laboratory and the intratracheal injection control did not show evidence of proteinosis.

 

Intratracheal Instillation:

Lung histology

Rats:

Pyro and atomized powder - 100 mg/m³

6 mths: numerous large foci of collagenous fibrosis “sharply circumscribed but highly irregular in outline”; some coalesced; no remaining alveolar structure; coarse bundles of collagen; moderate number of plump connective cells; black pigment masses in connective tissue; alveolar tissue between fibrotic foci usually normal.

12mths: collagenous foci with more fibres and fewer connective cells; similar between the different powders; inter-animal variability in response was evident.

Pyro and atomized powder – 12 to ≤24 mg/m³

Smaller, more widely separate foci that were highly cellular with only a few collagen fibres; foci were concentrated around the respiratory bronchioles and alveolar ducts.

Pyro and atomized powder – ≤12 mg/m³

No significant collagenisation of foci at 6 or 12 mths.

 

Conclusions:
Intratracheal injection of aluminium powder 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. There was no dose response evident or a noticeable difference between responses to the different aluminium powders.

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 actual inhalation exposures. All three species developed widespread alveolar proteinosis, rats exhibiting the most severe response. The proteinosis resolved progressively after cessation of exposure. The group of rats exposed for 12 months to 15 mg/m³ of aluminium powder showed moderate alveolar proteinosis after only 6 months of exposure.
Executive summary:

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 75 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 75 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 1mL 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 hematoxylin-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 15mg/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% <1μm) included in the study as a “non-fibrogenic” control.   

The reason for the high and variable rates of mortality in this study is unclear and is a limitation of the study. Several endpoints specified in the chronic toxicity guideline (OECD TG 452) were not assessed, particularly body and organ weights. The study design and animal husbandry were not described in sufficient detail. Considering reliability for use in the hazard identification, a Klimisch Score of 2 is appropriate for the lung pathology results and a Score of 3 for the mortality results.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
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:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
: Only one sex of animals; Number of animals per group (sex/dose/timepoint); Outcomes assessed (lack of observations of body weight and other clinical signs); lack of information on animal husbandry
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Details on test animals and environmental conditions: no data
It is unclear whether the animals remained in the inhalation chambers even when the exposure was not occurring.

Diet and water: NR

Acclimation and monitoring animal health:
No information was provided on acclimation or animal care.

Route of administration:
other: Inhalation: dust and Intratracheal injections
Type of inhalation exposure:
whole body
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: No information was provided on the MMAD and GSD.

Further detail on particle characteristics (e.g. shape):
Particle size by count was provided for the size ranges < 1.0 µm; 1 to 4 µm; and > 4 µm.
(1) British pyro powder
Shape: flake-like
Size: < 1 µm 4.2%, 1 – 4 µm 87.3%, > 4 µm 8.5%
Mean diameter: 2.49 µm
Specific surface area: 10.4 m²/g

(2) US –source atomised particles.
Shape: “spherical”
Size: < 1 µm 1.5%, 1 – 4 µm 95.6%, > 4 µm 2.9%;
Mean diameter: 2.22 µm
Specific surface area: 0.8 m²/g

(3) US-source flake powder
Shape: flake
Size: < 1 µm 0.0%, 1 – 4 µm 28.6%, > 4 µm 71.4%;
Mean diameter: 4.85 µm
Specific surface area: 8.4 m²/g

(4) Negative control: aluminium oxide dust.
Shape: not stated
Size: < 1 µm 66%, 1 – 4 µm 25%, > 4 µm 9%;
Mean diameter: 0.80 µm
Specific surface area: 6.3 m²/g
Details on inhalation exposure:
Further details on inhalation exposure:
The chambers were approximately 1.2 m³ in volume. Moisture was removed using anhydrous calcium chloride. Powders were dispersed through the chambers by means of a dust-feed mechanism (Wright). Air flow was limited to 10 litres/min to attain high dust concentrations.

Details on Intratracheal Instillation:
A suspension of the dust in tap water was instilled intratracheally. 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.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The air flow through the chambers was monitored with outflowing chamber air forced through 20.3 x 25.4 cm filter (Millipore) filters before ventilation. The filters were used to gravimetrically estimate the average dust concentration in the chamber each day. The data were not reported however.
Duration of treatment / exposure:
Inhalation
Rats in the 50 and 100 mg/m³ chambers were exposed for 6 months. Exposure duration was 12 months for the animals at the lower aluminium powder concentrations of 15 and 30 mg/m³.

The aluminium oxide control rats were exposed to 75 mg/m³ for six months. An additional 30 rats and 12 guinea pigs were exposed to 30 mg/m³ of aluminium oxide dust for a year.
Frequency of treatment:
Inhalation
6 hr/day; 5 days a week
Dose / conc.:
15 mg/m³ air
Remarks:
pyro powder, atomized powder, flake powder
Dose / conc.:
30 mg/m³ air
Remarks:
pyro powder, atomized powder, flake powder
Dose / conc.:
50 mg/m³ air
Remarks:
pyro powder, atomized powder
Dose / conc.:
100 mg/m³ air
Remarks:
pyro powder, atomized powder
Dose / conc.:
30 mg/m³ air
Remarks:
aluminium oxide
Dose / conc.:
75 mg/m³ air
Remarks:
aluminium oxide
No. of animals per sex per dose:
Inhalation
Rats:
30 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
30 rats were exposed to aluminium oxide dust at 30 and 75 mg/m³

- 5 animals were sacrificed per time point (6, 8, 12 and 18 months).

Intratracheal instillation **
15 rats 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.

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 were sacrificed at 2 months, 3 at 4 months and 2 at 6 months.
Control animals:
yes
Details on study design:
Control animals:
50 rats were untreated (laboratory controls). Five animals were examined per time point.
Aluminium oxide (numbers and dosing described above) were included as “non-fibrogenic” controls.
For the intratracheal instillation group, 15 rats were included as vehicle controls.

No information was provided on the method used to allocate the animals to groups.
Positive control:
No.
Observations and examinations performed and frequency:
Observations and examinations performed:
No information was provided on observations to monitor animal health but mortality was recorded.

Frequency of the observations and examinations:
For the inhalation exposure, pathological examinations took place at 6, 8, 12 and 18 months into the experiment for the 50 and 100 mg/m³ aluminium powder dose groups and the 70 mg/m³ aluminium oxide dose group (i.e. 0, 2, 6 and 12 months after cessation of exposure). Kills of the lower dose animals took place at 6 and 12 months (0 and 6 months post-exposure).
For the intratracheal instillation, see ** above.
Sacrifice and pathology:
The method used to sacrifice the animals was not reported in the article.
Histopathological examinations of lung tissue were conducted using sections cut in triplicate and embedded in paraffin blocks. One section was stained with eosin to show aluminium particles, a second section was stained with hematoxylin-eosin, and a third section with PAS or van Gieson. To show cellular components and stromal support structures, the hematoxylin-eosin stained sections were photographed then decolorized and impregnated with silver (Gordon and Sweets method) before another photograph was taken. Aluminium particles were removed prior to this procedure using 10% sodium bisulfite.
Other examinations:
No data.
Statistics:
No information was provided on statistical methods used for comparing mortality rates.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
A higher rate than ideal was observed. As high rates were observed in control groups as well as treated groups and appeared to show no relationship to dose, deaths were unlikely to be simply related to the dust exposure. The authors suggest an effect of crowding and low air flow in the chambers. Quantitatively, the mortality results from this study are not reliable. The absence of observations of clinical signs also suggests that caution ought to be used in interpreting the results.

ORGAN WEIGHTS
Lung weights were measured but not reported.

HISTOPATHOLOGY: NON-NEOPLASTIC
ADEQUATE
- alveolar proteinosis was observed after 6 months exposure to 15 mg/m³ in rats.
- foci of fibrosis found for pyro Al powder at 50 mg/m³ exposed for 108 days; killed 6 months later.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Pulmonary lymphoid tumors, reticulum cell and lymphosarcoma were noted in both the experimental and control groups. These were interpreted as spontaneous tumors in aging rats not associated with pulmonary dust exposure.




Dose descriptor:
LOAEC
Remarks:
local effects
Effect level:
15 mg/m³ air
Based on:
test mat.
Remarks:
Al powder
Sex:
not specified
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
>= 75 mg/m³ air
Based on:
test mat.
Remarks:
Al2O3 dust
Sex:
not specified
Basis for effect level:
other: no adverse effect observed at the highest dose tested
Critical effects observed:
not specified

Inhalation series:

Mortality: 

Spontaneous deaths were more numerous among all 3 species than ideal. The % of the animals dead at 6 months and 12 months are provided in the table below. The numbers are extracted from Tables 3, 4 and 5 of the publication.

 

Animal

Dust Type

Dose (mg/m³)

Exposure duration

% dead: 6 mos.

% dead: 12 mos.

Rats

Atomised Al

100

6 mos

0

0

 

Atomised Al

50

6 mos

7

25

 

Atomised Al

30

12 mos

0

28

 

Atomised Al

15

12 mos

0

8

 

Pyro Al

100

6 mos

0

40

 

Pyro Al

50

6 mos

0

20

 

Pyro Al

30

12 mos

0

20

 

Pyro Al

15

12 mos

3

36

 

Flake Al

30

12 mos

0

24

 

Flake Al

15

12 mos

0

32

 

Al2O3

75

6 mos

0

0

 

Al2O3

30

12 mos

0

20

Air control

0

6 mos

0

0

Air control

0

12 mos

0

0

 

 

Lung histology

Al-powders:

All three species developed alveolar proteinosis (AP);

 

Rats:

50 and 100 mg/m³ exposed for 6 mths:

Marked AP; but alveolar walls were generally thin and appeared normal;

AP underwent spontaneous resolution with little evidence remaining 1.5 years post-exposure.

15 and 30 mg/m³ for 12 mths:

Moderate AP from 6 to 12 months followed by gradual clearing. Some AP still present at 24 mths.

 

Persistent changes:

Small scattered foci of endogenous lipid pneumonitis (granulomatous inflammation) associated with cholesterol crystals that were not surrounded by AP material. These occurred generally not in regions with dust particles. The foci left collagenous scars.

No carcinoma was observed. Lymphoid tumors, reticulum cell and lymphosarcoma noted in both the treated and control groups. Considered spontaneous by authors and numbers were not provided.

 

 

Al2O3:

Rats:

Small foci concentrated in respiratory bronchioles and alveolar ducts – consisting of clustered alveoli with swollen macrophages engorged with particles; no thickening of alveolar walls evident; no evidence of AP or pnuemonitis.

 

Distribution and clearance of dust:

Dust remained finely dispersed even within the cytoplasm of macrophages.

Rats:

50 and 100 mg/m³ exposed for 6 mths: Clearance by 1.5 years post-exposure

15 and 30 mg/m³ exposed for 12 mths: some finely dispersed Al-powder particles were still evident 1 year post-exposure.

 

There was no dose response evident or noticeable differences in response to the different aluminium powders.

 

The laboratory and the intratracheal injection control did not show evidence of proteinosis.

 

Intratracheal Instillation:

Lung histology

Rats:

Pyro and atomized powder - 100 mg/m³

6 mths: numerous large foci of collagenous fibrosis “sharply circumscribed but highly irregular in outline”; some coalesced; no remaining alveolar structure; coarse bundles of collagen; moderate number of plump connective cells; black pigment masses in connective tissue; alveolar tissue between fibrotic foci usually normal.

12mths: collagenous foci with more fibres and fewer connective cells; similar between the different powders; inter-animal variability in response was evident.

Pyro and atomized powder – 12 to ≤24 mg/m³

Smaller, more widely separate foci that were highly cellular with only a few collagen fibres; foci were concentrated around the respiratory bronchioles and alveolar ducts.

Pyro and atomized powder – ≤12 mg/m³

No significant collagenisation of foci at 6 or 12 mths.

 

Conclusions:
Intratracheal injection of aluminium powder 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. There was no dose response evident or a noticeable difference between responses to the different aluminium powders.

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 actual inhalation exposures. All three species developed widespread alveolar proteinosis, rats exhibiting the most severe response. The proteinosis resolved progressively after cessation of exposure. The group of rats exposed for 12 months to 15 mg/m³ of aluminium powder showed moderate alveolar proteinosis after only 6 months of exposure.
Executive summary:

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 75 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 75 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 1mL 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 hematoxylin-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 15mg/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% <1μm) included in the study as a “non-fibrogenic” control.   

The reason for the high and variable rates of mortality in this study is unclear and is a limitation of the study. Several endpoints specified in the chronic toxicity guideline (OECD TG 452) were not assessed, particularly body and organ weights. The study design and animal husbandry were not described in sufficient detail. Considering reliability for use in the hazard identification, a Klimisch Score of 2 is appropriate for the lung pathology results and a Score of 3 for the mortality results.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
75 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

There are no studies available on the repeated dose toxicity of aluminium oxide by the oral route.

In terms of hazard assessment of toxic effects, available data on the repeated dose toxicity of other aluminium compounds was taken into account by read-across following a structural analogue approach, 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).

A detailed rationale and justification for the analogue read-across approach is provided in the technical dossier (see IUCLID section 13).

Oral:

In terms of hazard assessment of toxic effects, available data on the repeated dose toxicity of other aluminium compounds was taken into account by read-across following a structural analogue approach, 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).

A GLP study was performed using aluminium chloride basic in accordance with OECD Test Guideline (TG) 422 "Combined Repeated Dose and Reproductive/Developmental Screening Test" (Beekhuijzen 2007). No mortality or clinical signs of intoxication were observed in male and female Wistar rats due to treatment with Al chloride basic at dose levels of 40, 200, and 1000 mg/kg body weight which contribute 0, 7.2, 36 and 180 mg Al/kg bw/day, respectively.

Treatment with Al chloride basic by oral gavage revealed paternal toxicity (irritation effect on glandular stomach mucosa, local effect) at 1000 mg/kg in both the male and female Wistar rats. Based on findings observed macroscopically (red foci or thickening of the grandular mucosa of the stomach) and supported by microscopic examination, the maternal/parental No Observed Adverse Effect Level (NOAEL) for local toxic effects on stomach was established at 200 mg/kg and LOAEL – at level 1000 mg/kg, for both males and females.

Several statistically significant changes in clinical biochemistry parameters were observed at 1000 mg/kg: decreased Hb level in males, MCHC in both Al treated males and females, decreased alkaline phosphatase activity, decreased total protein and albumin levels in blood serum and increased potassium level. Decreased Hb levels were observed in two other doses in males but no dose response relationship was observed. Lack of relevant baseline values for the observed clinical data limit the interpretation of the results.The authors consider clinical biochemistry and haematology changes observed at 1000 mg/kg to be of slight nature and generally within the range expected for rats of this age and strain.Because any morphological correlates were absent, these changes were considered not indicative of organ dysfunction and not of toxicological significance.

No reproduction, breeding and early post-natal developmental toxicity was observed in rats at 1000 mg/kg body weight for males and females. Based on the reported results, a NOAEL for reproduction, breeding and early post-natal developmental toxicity was suggested at a level of 1000 mg/kg bw, the highest dose tested in this study.

 

In another study (Hicks et al., 1987) Male Sprague-Dawley rats were exposed to aluminium-compounds with diet. The animals were randomly assigned to five groups, 25 animals in each. The groups received 1) basal diet (control), 2) aluminium hydroxide (302 mg Al/kg body weight), 3) KASAL -the basic form of sodium aluminium phosphate containing ≈6% of Al (141 mg Al/kg body weight), 4) KASAL II - the basic form of sodium aluminium phosphate containing ≈13% of Al (67 mg Al/kg body weight) and 5) KASAL II (288 mg Al/kg body weight). Treatment continued for 28 days, during which the animals were observed twice daily for their behaviour, signs of toxicity, and mortality. General physical examinations, body weight and food consumption measurements were performed weekly. After 28 days of treatment, 15 animals from each group were killed. Blood was collected from 5 rats of each group for blood cell counts, haemoglobin concentration, haematocrit and serum chemistry measurements. These rats were subjected to gross necropsy and histopathological examination. Femurs from 10 rats were taken for possible aluminium analysis; femurs from 5 rats were analyzed for Al concentrations. Five rats were allowed to recover for 2 months and five rats for 5 months after termination of the treatment. During these recovery periods, the rats received the basal diet and were observed daily; body weight and food consumption were measured monthly. Femurs were collected at autopsy from these rats for aluminium analysis. During the entire experimental period, no mortality was reported and no treatment-related clinical signs were observed. All clinical observations were characteristic of male Sprague-Dawley rats of relevant age. There were no significant group differences in body weight, food and water consumption and haematological parameters.A mild (2 - 4%) but significant increase in serum sodium level was observed in all treated animals.However, all increased sodium levels were within the range of historical control for rats of the same age in the laboratory. A significant 16% increase in absolute kidney weight was reported in the group of rats receiving KASAL II at 67 mg Al/kg bw. This increase appeared not to be treatment-related because no such increase was seen in the group of animals treated with this substance at 288 mg Al/kg bw. There were no other significant group differences in organ weights. All lesions seen at microscopic examination were “those normally expected for young adult male Sprague-Dawley rats” (Hicks et al., 1987). No lesions suggestive of a treatment-related effect were seen.Aluminiumconcentrations in all femur samples from all groups were < 1 ppm and most were below the limit of detection or quantification.The distribution of samples in which Al was not detectable, was detectable but not quantifiable or was quantifiable, was similar in all the groups. It should be noted that this comparison was based on small numbers of samples from each group (5). Al was quantifiable in all 5 samples from animals treated with Al(OH)3, in 2 samples from the control animals and in none of the samples from animals treated with KASAL or KASAL II. The results of this study provide no evidence for significant deposition of Al in the bone and no evidence for adverse effects induced by Al hydroxide or basic food grade sodium aluminium phosphate (KASAL and KASAL II) during 28-day dietary administration at daily doses up to ≈300 mg Al/kg body weight. 

 

Sodium aluminium phosphate was administered to beagle dogs with diet at concentrations 0% (control), 0.3%, 1.0% and 3.0% for 6 months (Katz et al., 1984). There were no significant group differences in body weight throughout the experiment. Reductions in mean body weight occurred in all groups during week 27, which the authors attributed to “pretermination tests and increased handling by technicians.” No treatment-related clinical signs and no ocular changes in any of the animals were observed. In most weeks, treated male and female dogs consumed less food than control dogs. In male animals, none of the differences in mean food consumption values was statistically significant. In females, significant reductions occurred “sporadically”. The authors did not consider these differences in food consumption as “toxicologically significant”, the conclusion that was supported by the absence of corresponding reduction in body weights. The treatment did not have any effect on haematological and blood biochemistry parameters, urinalysis results and results of analysis for occult blood in faeces. There were no significant differences in mean organ weights between the treated groups and the control group. Gross pathology and histopathology findings were in the “normal range of variations for dogs of this strain and age”; no treatment-related lesions were observed. The results of this study provide no evidence for toxicity of acidic form of sodium aluminium phosphate during 6-month administration at concentrations up to 3% in the diet.

 

Aluminium citrate was administered to ten female Sprague Dawley rats with drinking water at a concentration of 80 mmol/L for 8 months (equivalent of 6 - 11 mmol Al/kg bw/day) (Vittori et al. 1999). Plasma iron concentration and total iron-binding capacity were not different in the control and the Al treated rats, indicating that the Al treated animals were not depleted of iron. There were no significant group differences in blood urea concentration, which suggests that kidney function was not altered by Al administration. Significantly lower haematocrit and blood Hb concentration were observed in the Al treated rats than in the control rats. Significantly higher reticulocyte count, abnormal erythrocyte morphology, a significant inhibition of (late colony-forming unit-rethroid, CFU-E) growth and a significant reduction of 59Fe uptake in the bone marrow were reported in the Al treated rats. Plasma haptoglobin concentration was significantly lower in the Al treated animals than in the control animals. This and the presence of abnormal erythrocytes in the Al treated rats are indicative of intravascular haemolysis. Scanning electron microscopy combined with EDAX detected Al inside circulating erythrocytes with abnormal shape from animals in the Al treated group. Al concentrations in the bone, spleen, liver, kidney and plasma were significantly higher in the Al treated group than in the control group. No significant group difference in brain Al concentrations was seen. There was no correlation between plasma Al concentrations and Al levels in the organs or any other biochemical data. The results of this study suggest that Al may affect erythropoiesis in rats with normal renal function.

 

A recent combined one-year developmental and chronic neurotoxicity study with Al-citrate (Alberta Research Council Inc, 2010) may be of interest for the evaluation of the neurotoxicity of aluminium hydroxide, aluminium metal and aluminium oxide taking into consideration the tenfold lower bioavailability of aluminium hydroxide, aluminium metal and aluminium oxide compared to Al-citrate and excluding effects that can likely be related to the salt rather than the cation. The study was conducted according to OECD TG 426 and GLP, and the exposure covered the period from gestation day 6, lactation and up to 1 year of age of the offspring. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions  via drinking water of  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). The highest dose was a saturated solution of Al-citrate. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L, equimolar in citrate to the high dose Al-citrate group) 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. Dams were sacrificed at PND 23. At PND 4  1 male and 1 female pup of each litter  were allocated to 4 testing groups: D23-sacrifice group for pre-weaning observations and D23 neuropathology, D64, D120 and D365 postweaning groups for post weaning observations and neuropathology at the respective days of sacrifice. Endpoints and observations in the dams included water consumption, body weight, morbidity and mortality and a Functional Observational Battery (FOB) (GD 3 and 10, PND 3 and 10). Pups were examined daily for morbidity and mortality. Additional neurobehavioral tests were performed at specified intervals and included, T-maze, Morris water maze, auditory startle, and motor activity. Female pups were monitored from PND26 for vaginal opening, male pups from day 35 for preputial separation. Clinical chemical and haematological analysis was performed for each group on the day of scheduled sacrifice. Al-concentrations were determined in blood, brain, liver, kidney, bone and spinal cord tissues by inductively coupled plasma mass spectrometric analysis. Further metals such as iron, manganese, copper and zinc were also determined. The pathological investigation includes rain weight and neuropathology. Statistical analyses were performed using the SAS software release 9.1. Data collected on dams and pups were analysed separately. All analysis on pups was performed separately for each sex. Statistical significance was declared from P ≤ 0.05.

Results: Dams: Eight high dose dams developed diarrhoea. In the Na-citrate group one dam stopped nursing and the pups were euthanized. No significant differences between mean body weights of dosed animals compared to controls were observed during gestation and lactation. During gestation and lactation low and mid dose group animals consumed considerably more fluid than controls and high dose group animals. This is not considered treatment related as there was no dose response. In all animals the target dose was exceeded during lactation due to the physiologically increased fluid consumption.

Pups: During the pre-weaning phase weights of mean body weights of male and females in the sodium citrate and high dose group were significantly lower than the untreated controls. This suggests a citrate rather than Al-related effect. No differences between treated and control animals were observed in the FOB. No other clearly treatment related effects were observed pre-weaning.

F1-postweaning: General toxicity

No significant differences in body weights throughout the study were observed between low and mid-dose animals sodium-citrate and untreated controls. High dose males had significant lower body weights than controls by PND 84. These animals also had clinical signs. At necropsy urinary tract lesions were observed in the animals of the high dose group, most pronounced in the males, hydronephrosis, uretal dilatation, obstruction and/or presence of calculi. All high dose males were sacrificed on study day 98. The effect is probably due to Al-citrate calculi precipitating in the urinary tract at this high dose level. This effect is related to the citrate salt and cannot be attributed to the Al-ion. Female high dose animals showed similar urinary tract lesions, but with a lower incidence and severity. Urinary tract lesions were also observed in single mid dose males, but also in a few sodium citrate and control animals. Fluid consumption during the study was increased in the sodium citrate and Al-citrate groups (in particular high and mid dose) compared to controls. This is probably due to the high osmolarity of the dosing solutions. However, the consumed dose levels decreased in all dose groups during the study. In the beginning the target dose was considerably exceeded, while versus the end of the study it was considerably below the target dose.  According to the authors the assigned dose levels still remain valid.

Developmental landmarks:

In sodium citrate controls and high dose males and females the number of days to reach preputial separation or vaginal opening was longer than in untreated control animals. This may be related to the lower body weights in these animals at the respective time-point. As the sodium citrate group showed similar retardation this effect cannot be allocated to the aluminium cation.

Neurobehavioral testing

No consistent treatment related effects that could be related to Al-ion exposure were observed in the FOB. No treatment related effects on autonomic or sensimotoric function were observed in the study. A weak association between Al exposure and reduced home cage activity, a very weak association with excitability, some association with neuromuscular performance were reported but according to the authors this may also be related to group differences in body weight, and an association with physiological function and is thus not considered clearly treatment related. No treatment related effect on general motor behavior was observed. No clearly treatment related effect on auditory startle response was observed. There was no evidence of any treatment related effect on learning and memory in the Morris Water Maze test and no clearly treatment related effects in the T-maze test. Hind limb grip strength and to a lesser extend foot splay were reported to be reduced compared to controls in high and mid dose male and female animals, more pronounced in younger than in older  rats. However, the observed effects can be related to the lower body weights of the individual animals undergoing this test. No details on the individual findings and historical control data are available. It can therefore not be concluded with certainty that the observed neuromuscular effects are primary effects of the treatment and attributable to Al3+. The NOAEL was reported based on this effect as 30 mgAl/kg bw in a conservative approach.

Haematology: No clinically significant differences in hematology were observed at the investigation on day 23. In day 64 and 120 females and day 64 males the high dose group showed slight reduction in hematocrit (males only), mean hemoglobin and mean corpuscular cell volume. No such changes were observed in the 364 day group.

Clinical chemistry: while a number of borderline statistically significant changes were observed, such as globuline levels, alkaline phosphatase and glucose in the high dose group little or no biological significance is associated with them. Elevated creatinine and urea levels in Day 64 males are consistent with the renal toxicity observed in these animals.

Organ weights: Brain weights did not differ among the groups, with two exceptions in the day 64 group males brain weights were significantly lower than controls. In the 120 day female high dose group brain weights were also significantly lower than controls. These findings were not reproduced at the other sacrifice times. Brains to body weight ratios were not significantly different and the lower brain weights can be attributed to the body weight.

Pathology: The main pathology findings were the renal lesions with precipitates in the urinary tract and secondary lesions such as hydronephrosis and uretal dilatation   in particular in the high dose group males and to a lesser extend females. Fluid colonic content was also observed in some high dose animals, in particular males. According to the authors the test item clearly precipitated in the urinary tract causing stone formation and blockage and resulted in fluid colonic content. No other macroscopic effects were observed in other organs.

Histopathology: No treatment related histopahological effects were observed in the nervous system at any time point.

Aluminium concentrations in different organs were dose related. Tissue concentrations were highest in blood, and then in decreasing order brainstem, femur, spinal cord, cerebellum, liver cerebral cortex.

A conservative NOAEL of  322 mg Al-citrate/kg bw  corresponding to 30 mg Al/kg bw was derived from this study (with a bioavailability correction this would correspond to ca. 300 mg Al from Al(OH)3).

The most important effects were however related to a precipitation of the citrate in the kidneys and urinary tract and this effect is not related to the Al3+ ion.  The effects on grip strength and foor splay observed can also not be attributed unequivocally to Al-exposure as they may have been secondary to the general toxicity and body weight differences between treated and control animals undergoing this test.

 

 

 

Inhalation:

Human Studies

Aluminium powder

The majority of published human studies of lung effects on exposure to aluminium powder were conducted prior to 1970 (Krewski et al., 2007 (review); Doese, 1938; Goralewski, 1939 to 1948 in Perry, 1947; Koelsch, 1942; Meyer and Kasper, 1942a,b; Crombie et al., 1944; Mitchell et al., 1959, Mitchell et al., 1961; McLaughlin et al., 1962). Effects were associated with intermittent use of a more permeable and possibly biologically active petroleum-based mineral oil coating in place of stearine (Dinman et al., 1987). The small, cross-sectional study (n = 62) by Kraus et al. (2006) provides some evidence for the development of lung pathology (small, round opacities in the upper lung; a thickening of the interlobular septae) consistent with alveolitis without fibrotic activity on exposure to aluminium powder (respirable size range; with diameters smaller than 5μm). Exposure duration in this study ranged from 78 to 360 months. Multivariate logistic regression showed a significant independent association between Al levels in urine and the occurrence of abnormal high resolution computed tomography (HRCT) findings (OR = 1.008, 1.002 - 1.013; 95% CI; p < 0.006; with adjustment for age, time of exposure, smoking habits, vital capacity, FEV1/VC, and resistance). 

 

“Aluminium” dust

Miller et al.(1984) observed pulmonary alveolar proteinosis in a 44-year old male who had been exposed to high levels of aluminium-containing dust during 6 years as a rail grinder. A recent report (Cai et al., 2007) reported granulomatosis lung disease in a 50-year old woman who had worked in a metal reclamation factory and been exposed to high levels of aluminium dust. Energy dispersive X-ray analysis of the granuloma tissue showed high concentrations of aluminium. Separation of an effect specific to aluminium from an effect due to high doses of dust, or in fact, aluminium oxide, is not possible based on these studies.

 

 

Aluminium smelters - occupational asthma

Donoghue et al. (2010) studied occupational asthma among employees in Al pre-bake smelters of Australia and New Zealand from 1991 to 2006 and examined relations between asthma in highly exposed workers and potroom air contaminants. The authors collected asthma incidence each year by a survey of seven Al smelters using diagnostic criteria developed in 1990 by the Australian Aluminium Council Health Panel. Regular medical surveillance, including respiratory questionnaires and spirometry, was conducted at all smelters with intervals from 3 months to 2 years between examinations depending upon job type and duration of employment. No information was available on ages of the workers, gender or range of length of employment. Asthma cases were identified by surveillance following development of symptoms or a few of the cases were diagnosed by a family physician. Pre-placement criteria and assessment of individual suitability for jobs with exposure to potroom dust, fumes, and gases were introduced before the study period; these criteria evolved over the course of the study and these criteria were not uniform at all smelters. These parameters included a history of asthma beyond childhood, reduced forced expiratory ratio (FER) and evidence of reversible airway obstruction. In some smelters, assessment of non-specific bronchial hyper-responsiveness using methacholine challenge was performed. Incidence rates for occupational asthma were calculated for each smelter and for all smelters combined and the data were presented for each year of the study. All cases of occupational asthma identified among smelter employees (regardless of job category) were divided by the total number of smelter employees (regardless of job category) and the incidence rates were expressed as the number of cases per 1,000 employees per year. These annual surveys also obtained data on the work areas in which asthma cases were reported, but due to limited data on employees in each work area, incidence rates by work area were not calculated. Employees who worked ‘‘in close proximity to pot fume or bath material for several hours a week as part of their normal job” (e.g., potrooms, potroom services, rodding, potlining, cryolite recovery, scrubbing, and alumina) were defined as the highly “bath exposed” workers. Exposure data were based on personal sampling of inhalable particulate, respirable particulate, particulate fluoride (F), gaseous hydrogen fluoride (HF) and total F for potroom employees charged with anode changing as it was the most consistent job across all of the smelters. Exposure data were collected from the breathing zone of potroom employees (the numbers of employees were not provided) under the supervision of qualified occupational hygienists for each year (1996 – 2006), but the study design was such that use of personal respiratory protection was not taken into account.

The statistical significance of changes in exposure concentrations (mg/m³) of inhalable particulate, respirable particulate, particulate fluoride, gaseous hydrogen fluoride, total fluoride across all Al smelters during the study period was assessed by regression P-values and Spearman’s correlation coefficients were calculated for correlations between the incidence rate and each exposure variable. A total of 329 cases of occupational asthma were identified and the highest rate occurred in 1992 (9.46/1,000 per year), but this declined to 0.36/1,000 per year in 2006. This amounted to a 96.2% reduction in asthma incidence. Of the 329 cases, 180 (55%) occurred in potroom production employees and of the total at least 243 of those cases (74%) occurred in employees who were assigned duties in the ‘‘bath exposed’’ areas.The mean proportion of all employees who were ‘‘bath exposed’’ over the period 1991 – 2006 was 50% (2,916/5,827) (no further details provided). The median values of the geometric mean concentrations of inhalable particulate, respirable particulate, particulate F, gaseous HF and total F across all seven of the Australian and New Zealand smelters in the worker’s breathing zone declined over the study period. Statistically significant correlations were observed between the reductions in the incidence rate of asthma and reductions in total respirable particulate, total F, particulate F and gaseous HF. The correlation coefficient was greatest for total F (rs= 0.497).

The Donoghue et al. (2010) results demonstrate reductions in occupational asthma among employees of seven New Zealand and Australian Al smelters from 9.46 per 1000 employees per year in 1991 to 0.36 per 1000 employees per year in 2006. Moreover, this reduction was correlated with reductions in the geometric mean of total F in the breathing zone among employees undertaking anode changing (rs = 0.497, p < 0.001). 

A number of the potroom exposure control measures were implemented during the study period (1991 - 2006) and these included an increased focus on standardized work practices, exposure monitoring programs were improved, hooding of pots was performed, ore and fluoride delivery systems were enclosed and enclosed overhead crane cabins with air purification were added and quality control on anode manufacture was improved to reduce replacement of failures. In addition, crucible cleaning and maintenance operations were isolated from potrooms, anode butt cooling was conducted in areas remote from the main potroom aisleway (a practice employed in only some smelters), exhaust ventilation of pots and forced draft ventilation were added in some smelters, natural dilution ventilation of potrooms was augmented by automated fume control in some smelters, an increased focus on consistent alumina/bath anode covers with no holes in the crust, improved process control to minimize anode effects, reduced process upsets that required intervention, improved fume system maintenance to minimize fume system outages, real-time gaseous HF fluoride monitoring in potroom roves with warning signals, controlled sweeping for fugitive dust on floors (e.g., prohibitions on compressed air for sweeping), automated anode butt cleaning and an increased focus on housekeeping. Among the improvements were: additional education on potential health impacts and work practices to minimize exposures, mandatory respiratory protection with clear rules, introduction of enhanced respirator selection and use of powered air purifying respirators (PAPR), quantitative respirator fit testing, education on respirator use, dedicated respirator maintenance and cleaning centers in some smelters and biological monitoring of urinary F for assessment of respiratory protection in some smelters. The Al smelting process was improved over time by blending low sulfur coke with normal coke to reduce SO2 emissions.

There is no question that misclassification of workers can influence the outcome of these types of studies be they prospective or retrospective in nature. Concern can also arise regarding comparisons based on incomplete or highly variable exposures within jobs or between all workers with asthma. If in these circumstances only “bath exposed” workers are considered, this selection can introduce non-differential misclassification of equal or perhaps even greater gravity to the use of area sampling data or failure to measure workplace levels at all.

Fluoride exposure has previously been associated with development of asthma symptoms and non-specific bronchial hyper- responsiveness in cohort studies of Norwegian potroom workers (Kongerud et al., 1991; 1994; Søyseth et al., 1994). However, occupational exposure data for these older studies were incomplete. The strongest epidemiological evidence is likely to come from an inception cohort study of new workers with detailed longitudinal characterization of personal exposures to the host of airborne materials found in Al smelters (Abramson et al., 1989).

Strengths of the Donoghue et al. (2010) study design include: consistent diagnostic criteria that were applied throughout the study period, prospective collection of asthma incidence and collection of personal samples for the highly exposed workers. While the results are most encouraging, the study does suffer from a number of limitations including: data on cases of occupational asthma come from different sources (onsite medical centers and family physicians and it is not clear whether family physicians applied the same criteria to diagnose occupational asthma); the proportion of cases diagnosed by family physicians is not reported; missing data on numbers of asthma cases and/or numbers of employees for some years; the pre-placement criteria were different at different smelters and “evolved during the study period”; the pre-placement criteria applied only to jobs with highest potential for potroom exposures but the incidence rates of occupational asthma were calculated for all employees regardless of job category; there was no description of date(s) when pre-placement examinations were introduced in smelters and the possible impact of worker reassignment, migration out or replacement of the “bath-exposed” workers on the incidence rate was not discussed; lack of data on potential confounding factors (employee turnover rates, age, tobacco consumption). Although it is not clearly stated whether the mean Al exposures for anode changers represent full-time shift measurements or whether it represents the highest short-term or transient peak exposures to dust and gas, the values are likely to be 8-hour time-weighted-averages (the usual way in which occupational exposures of this type are reported). There may be concern regarding correlations between exposure among the most highly exposed employees and the overall rate of occupational asthma given that no data on the asthma rates among the highly exposed workers were presented. The limitations in exposure measures and failure to account for worker migration out of the industry provide the basis for a Reliability Score of 2. In any case no conclusion on a possible effect of Al-exposure can be drawn from this study.

 

Aluminum smelters exposure to airborne contaminants and respiratory outcomes  

Abramson et al. (2010) investigated the relationships between occupational exposures to airborne contaminants (total F, gaseous fluoride, sulphur dioxide (SO2), coal tar pitch volatiles (as benzene soluble fraction (BSF), oil mist and total inhalable dust) and changes in respiratory function over time. Following a cohort employed at two Australian Al smelters where 446 new employees (77% of the 583 eligible workers) were examined at regular intervals over a period of 9 years (from 1995 to 2003), all participants completed an interviewer-administered questionnaire, pulmonary function tests and skin prick testing for common aeroallergens. Most of the workers were under 35 years old, with a median age for men of 30 (IQR 23-36) years and women of 31 (IQR 25-37) years. At baseline interview, wheeze and chest tightness were reported by 22.6% and 10.6% participants, respectively, and 9.4% were diagnosed with asthma. Baseline pulmonary function findings were within normal values, but 57% were atopic on skin prick testing. At smelter A and smelter B, the proportion of current male tobacco smokers was 32% and 23%, respectively, and the proportions of current female smokers were 39% and 28%, respectively. The proportion of former (19%) and never smokers (51%) was similar between sites.

A task exposure database (TED) was used by site industrial hygienists to record routine air monitoring for the airborne fluorides, SO2, coal tar pitch volatiles (as BSF), oil mist and inhalable dust. Based on these data, a Task Exposure Matrix (TEM) was constructed for each contaminant for each full shift task at each site for each year of follow-up of the study (described in Benke et al., 2000). All individual exposures were assigned using the arithmetic mean for each job/task combination and those values were expressed in mg/m³. By combining the TEM with each worker’s job history, the cumulative exposure between interviews (years 3 mg/m3) was calculated for each constituent.

Data were analyzed with the Generalized Estimating Equations (GEE) method to account for the correlation among repeated measurements from each participant during follow up. Logistic models were used in analyses of health symptoms and BHR data and these models were adjusted for age, gender, age and gender interaction, tobacco smoking, smelter site, atopy and interaction between gender and smoking. Linear models were used in analyses of lung function and these models were also adjusted for age, gender, age and gender interaction, smoking, smelter site, height at entry interview and interaction between gender and smoking. Nearly all (98%) of the production, office (96%) and maintenance (95%) workers remained in the same group over the follow-up period. Cumulative exposures were described by tertile (but those data presented in the publisher’s appendix Table E2 were not available for review).

The highest prevalence and widest range of exposures were found for inhalable dust, but the empirical data were not presented. The 95thpercentiles for all airborne constituents considered in the Abramson et al. (2010) study were less than the current Australian Exposure Standards for an 8 h shift; only 1.1% of the cohort was exposed to total inhalable dust above the standard of 10 mg/m3. Asthma symptoms (wheeze and chest tightness) were associated with cumulative exposures to SO2 (p < 0.001 and p < 0.01, respectively), inhalable dust (p < 0.002 and p < 0.02) and coal tar pitch volatiles as the benzene soluble fraction (BSF) (p < 0.005 and p < 0.04). Fluoride (no details on the type of fluoride, e.g., gaseous or total) exposure was associated with wheeze (P < 0.04), but not with chest tightness.The authors reported that the association between wheeze and gaseous fluoride (OR 1.19, 95% CI 1.01 to 1.41 per tertile) was very similar to that for total fluoride (but those data were presented in publisher’s appendix Table E7 not available for review). When fluoride and inhalable dust were analyzed simultaneously for wheeze, only the coefficient for inhalable dust remained statistically significant (data from the publication referenced at appendix Table E4 were not available).Airflow limitation [the lower values of the forced expiratory ratio (FEV1/FVC)] was associated with higher cumulative exposures to coal tar pitch BSF (p < 0.03), fluoride (p < 0.04) and SO2 (p < 0.001). Longitudinal changes in pulmonary function (decline in FEV1 and FVC) were significantly associated with cumulative exposure to fluoride (p < 0.001 and p = 0.002, respectively), inhalable dust (p = 0.02 and p < 0.001, respectively) and SO2 (p = 0.001 and p = 0002, respectively).

Statistically significant associations were largely confined to male employees. The authors reported that the findings for gaseous F were similar to those for total F; however, data referenced at appendix Table E9 of the publication were not presented.The results suggested that the likelihood that bronchial hyper-responsiveness (BHR) increased significantly with cumulative exposure to coal tar pitch BSF (p = 0.03), fluoride (p = 0.03), inhalable dust (p = 0.001), SO2 (p = 0.009) and oil mist (p < 0.001).Bronchial hyper-responsiveness was associated with current tobacco smoking in females and atopy in both genders, but those data (referenced at publication appendix Table E11) were not presented. 

The strengths of the Abramson et al. (2010) study include the inception cohort design and robust assessment of workplace area exposures. The study is limited by possible misclassification of Al exposure including lack of personal sampling data (the results given in Benke et al., 2000; 2001). The data were expressed only as inhalable (total) dust and there was no differentiation between metallic Al and Al oxides. In addition, the fact data referenced in Tables E2-E11 were not available for review may limit interpretation of the results. The analyses were not adjusted for the use of personal protective equipment (PPE) and thus the reliability of the exposure data reported may be called into question. 

The Abramson et al. (2010) data establish relationships between cumulative occupational exposures to a number of airborne contaminants and occupational asthma among Al smelter workers. There was a significant association between chronic occupational SO2 exposure and wheeze and chest tightness, BHR reactions to methacholine challenge, reduced FEV1 and a longitudinal decline in lung function.A concentration-response relationship could be seen between fluoride exposure and those same outcomes, but the association was less evident. There was also a significant relationship between long-term cumulative exposure to inhalable dust and asthma-associated symptoms (wheeze and chest tightness), the longitudinal decline in pulmonary function (decline in FEV1 and FVC values) and increased BHR with the greater associations for wheeze and consecutive changes in FVC and BHR. Exposure to the BSF of coal tar pitch volatiles was also associated with increased asthma, airflow limitations and BHR, but coal tar pitch volatiles are not recognized as respiratory sensitizers. Oil mist exposure was also associated with increased BHR.Although many of the exposures were highly correlated, further statistical analyses suggested that of the known respiratory irritants, SO2 was more likely than fluoride to be responsible for many of the symptoms observed.In summary, chronic exposure to elevated levels of inhalable dust, SO2 and fluoride were the most important determinants associated with decrements in pulmonary function among these Al smelter employees. Again, no correlation to Al-levels was made and the study does not establish any relationship with Al-exposure.

Aluminum smelters - cardiopulmonary toxicity 

Friesen et al. (2010) studied acute and chronic polyaromatic hydrocarbon (PAH) exposure in relation to cardiopulmonary mortality in a cohort of 6,423 men and 603 women who worked for 3 or more years at an Al smelter in British Columbia, Canada. The authors linked data for the cohort to national mortality rates for the years 1957 to 1999 and examined exposure-response regarding the incidence of chronic respiratory diseases (COPD) and cerebrovascular disease. Work histories were abstracted from company records. Smoking status (ever smoking, never smoking, and unknown) was obtained through self-administered questionnaires sent to current workers, pensioners or to their survivors if the worker was deceased. Mortality of the cohort for select causes of death was compared with that of the whole of British Columbia using standardized mortality ratios (SMRs) adjusted for age, gender and time period. The development of a benzo(a)pyrene-based [B(a)P] quantitative job exposure matrix was described by Friesen et al. (2006). To create the [B(a)P] job-exposure matrix as a surrogate for total PAH exposures, statistical models were developed to derive annual arithmetic mean B(a)P levels for each operation and maintenance job in smelter potrooms that were based on personal exposure measurements collected from 1977 – 2000. This matrix accounted for different rates of exposure and declines in concentrations over time and potline. Exposure estimates for jobs without measurements were extrapolated from exposure estimates from the statistical models by adjusting for the amount of time worked in areas where ambient B(a)P levels had been determined.  Job- and time-period-specific B(a)P exposures levels were linked to each employee’s work history for calculation of cumulative and current B(a)P exposures. The models included smoking status and time-dependent covariates for years (5-year categories), time since first employed (years; continuous) and work status (employed at smelter: yes/no). For cerebrovascular disease and COPD, exposures were examined using cumulative B(a)P metrics (0-, 2-, 5-, and 10-year lags). The participants were workers who had a mean age of 32.4 years (range: 18–65), who were employed an average of 14.5 years (range: 3 – 45 years) and who contributed an average of 23.5 years (maximum 47) to study follow-up. Workers who died of ischemic heart disease (IHD) were more likely to have ever smoked than the average worker in the cohort (65% – 70% vs. 57%). The all-cause mortality SMR was less than that for the province’s population for both males (SMR = 0.87, 95% confidence interval (CI): 0.82 - 0.92) and females (SMR = 0.85, 95% CI: 0.63 - 1.11). Ischemic heart disease mortality (n = 281) was associated with cumulative historic B(a)P exposure (hazard ratio = 1.62, 95% confidence interval: 1.06, 2.46) in the highest category (> 66.7μg/m3-year). However, the higher hazard ratio for IHD found was for chronic B(a)P exposure and it was restricted to those who were in active employment (adjusted for smoking status and calendar year) - 2.39 (95% confidence interval: 0.95, 6.05) and who also had the highest cumulative B(a)P exposures (> 66.7μg/m3-year). The higher B(a)P exposures also had the widest confidence intervals. The stronger associations observed during employment suggest that cardiovascular effects may be reversible after termination of employment at the smelter even after adjusting for tobacco consumption. 

The Friesen et al. (2010) results suggest that cumulative workplace air PAH exposures in this Al smelter declined over time. One strength of the Friesen et al. (2010) study was its longitudinal design; however, the B(a)P exposures were not well characterized and exposure to Al and other constituents in smelter air was not taken into account. The authors elected to rely on mortality rates rather than morbidity for cardiopulmonary outcome and used semi-quantitative exposure estimates which may not reflect the actual PAH exposures. Failure to account for the host of factors known to influence cardiovascular health including diet, physical activity and the medical history of the participants are serious deficiencies. This study suggests there may be an association between heart disease and chronic PAH exposure in Al smelter workers. The study does not include an analysis of Al-exposure and related possible effects and is therefore not useful for the risk assessment of Aluminium.

 

Animal Studies

The high doses of particulates applied in animal studies tend to lead to overload-related effects (ATSDR, 2008; ILSI, 2000). Animal studies administering dust by intratracheal instillation (ITI) are not useful as sources of dose-descriptors for the inhalation route of exposure as some responses may result from the un-physiologic mode of administration. ITI studies can be useful, however, for screening and comparative ranking of particles for toxic effects. Several studies have shown interspecies differences in pulmonary reaction on exposure to aluminium metal and alumina (Engelbrecht et al., 1959; Gross et al., 1973; Christie et al., 1963).

Gross et al. (1973) did not observe development of alveolar proteinosis or thickening of alveolar walls in rats, hamsters or guinea pigs exposed to Al2O3 dust (66% < 1 μm). Pigott et al. (1981) reported no evidence of fibrosis in a repeated dose inhalation study that administered alumina fibres (Saffil) at levels between 2 and 3 mg/m³. The only pulmonary response observed was the occurrence of pigmented alveolar macrophages. 

Ess et al. (1993) investigated the subacute and chronic effects of short-term ITI administration (50 mg total dose, five 0.1 mL injections of suspension in sterile saline over a period of 2 weeks) of five smelter-grade and two laboratory-grade aluminas to Sprague-Dawley rats. These doses were sufficient to overload clearance mechanisms. All the dusts led to an inflammatory reaction in the alveoli evidenced through significantly elevated BALF total protein, LDH and sustained increases in PMN compared with the saline control. Only the laboratory grade aluminas showed signs of fibrosis (collagen) one year post-instillation, however.    

 

Adamcakova-Dodd et al. (2010) studied the pulmonary response following sub-acute inhalation exposure to aluminium nanowhiskers in mice. Aluminium nanowhiskers have been used in manufacturing processes as catalyst supports, flame retardants, adsorbents, or in ceramic, metal and plastic composite materials. Male mice (C57Bl/6J) were exposed to aluminium nanowhiskers for 4 h/day, 5 days/week for 2 or 4 weeks in the dynamic whole body inhalation exposure chamber. Control animals were exposed to a comparable sound level (80 dB) and laboratory air. The primary dimensions of these nanowhiskers were 2-4 nm x 2800 nm (Sigma-Aldrich) and the test nanomaterial [Al(OH)3:AlOOH] contained 35% Al.  The average concentration of aluminium nanowhiskers in the chamber was 3.3 ± 0.6 mg/m³ and median particle size diameter was 154.1 ± 1.6 nm. Both groups of mice were killed within 2 or 4 weeks of exposure at which time bronchoalveolar lavage (BAL) fluid was analyzed for differential and total cells, total protein, activity of lactate dehydrogenase (LDH) and cytokines. Total and differential white blood cells in the BAL fluid were counted. Lungs were processed for histopathology and pulmonary mechanics measurements were taken (flexiVent). The Al content in the lungs, heart, liver, spleen, kidney and brain was determined by ICP-OES. It was reported that the total number of cells as well as number of macrophages in BAL fluid was double that in mice exposed for 2 weeks and 6 times higher in mice exposed for 4 weeks, compared to air sham controls (p<0.01 and p<0.001, respectively). However, no neutrophilic inflammation in BAL fluid was found and the percentage of neutrophils was below 1% in all groups. No significant differences were found in total protein, activity of LDH, or cytokine levels (IL-6, IFN-γ, MIP-1α, TNF-α, and MIP-2) in BAL fluid between shams and Al-treated mice. In summary, sub-acute (2 or 4 weeks) inhalation exposures to Al whiskers increased the numbers of macrophages in BAL fluid. No other inflammatory or adverse responses were observed. Currently available information does not permit characterization of the nanowhiskers used in this study as particles or fibers.

This study provides new data on solubility and dissolution process of Al in biological fluids under different physiological conditions.However, the applicability of reported findings for manufactured Al2O3 nanomaterials to the bulk Al powders and dust is not clear.Only an abstract is available which limited interpretations of the study results. A Klimisch Score 4 (not assignable) was considered an appropriate for this study.

 

In vitro cytotoxicity studies

Aluminium metal reacts rapidly with air to form an aluminium oxide coat. Thus, exposure of tissues or cells to zero valence aluminium metal is unlikely by inhalation unless the aluminium powder is coated with a substance that acts as an effective barrier to oxidation in air but does not act as an effective barrier in the lung. The in-vitro studies by Wagner et al. (2007) and Braydich-Stolle et al. (2010) provide some evidence for a difference in cytotoxic effect between Al-NPs (2-3 nm oxide coat) compared with Al2O3-NPs. Al-NPs also showed an effect on macrophage phagocytosis of bacteria and particulates under the experimental conditions. The importance of this effect in-vivo in humans for larger particle sizes with thicker oxide coats is unclear. The utility of in-vitro studies for predicting the pulmonary toxicity profile in-vivo remains limited due to the dependence of biological effects, deposition, retention and inflammatory response on particle surface and physico-chemical characteristics (Sayes et al., 2007). 

 

Mechanism of Action

Overall, the results of the available in-vitro studies described earlier support the low cytotoxicity of poorly soluble aluminium oxide. Aluminium hydroxide and the closely related oxyhydroxide are similarly poorly soluble. These substances can be considered PSPs i.e. poorly soluble particulates of low cytotoxicity.

Summary

The current weight of evidence does not support a chemical-specific hazard on inhalation exposure to alumina (aluminium oxide, aluminium hydroxide) as experienced by the worker population. Gross et al. (1973) and Pauluhn (2009a) are considered the most adequate studies from which to obtain a dose descriptor to form the basis for a DNEL for repeated dose toxicity (inhalation, local effect) for these substances. The NOAEC from Gross et al. (1973), a subchronic study, for aluminium oxide (mean diameter 0.8 µm) is 75 mg/m³. The NOAEC from Pauluhn (2009a; sub-acute study; MMAD=1.7μm; agglomerated nanomaterials) for aluminium oxyhydroxide is 3 mg/m³ for a range of sensitive endpoints.

Considering aluminium oxide fume, the available information supports a low fibrogenicity (Stern and Pigott, 1983) and low cytotoxicity. 

 

Dermal

No animal studies are available in which the repeated exposure toxicity of aluminium has been investigated.


Justification for classification or non-classification

According to CLP (1272/2008/EC) classification criteria for repeated dose toxicity, no classification is required.