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Neurotoxicity

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Description of key information

A conservative NOAEL of 322 mg Al-citrate/kg bw/d for neuromuscular effects corresponding to 30 mg Al/kg bw/d was derived from this study. This would correspond to ca. 190 mg/ kg bw/d of aluminium sulfate as anhydrous form (molecular mass of 342,1 g/mol). 

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: oral
Remarks:
other: Developmental neurotoxicity and chronic toxicity study
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline with acceptable restrictions
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
other: OECD 426 and OECD 452
Deviations:
yes
Remarks:
: food consumption was not studied; exposure during in utero (GD 6-21) and weaning period (post-natal day (PND) 1-21), but the exposure of the rats to Al citrate continued beyond this period, until 12 months of age in one cohort
Principles of method if other than guideline:
Timed pregnant dams (n= 20 per group) were administered with aqueous solutions of aluminium citrate at 3 dosage levels (30, 100 and 300 mg Al/kg bw/day (nominal). Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L of citrate) or plain water (control group).
Al citrate was administered to dams via drinking water ad libitum from gestation day 6 through weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day 4. Offspring (female and male) were used to assess behavioral ontogeny, cognitive function, brain weight, clinical chemistry, hematology, tissue/blood levels of aluminium and neuropathology at various doses (30, 100 and 300 mg Al/kg) and time points (at PND 23 and 64, 120 and 364).
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 at study initiation: Females: 242.5 - 333.4 g (target 160-360 grams); Males: 335.4 - 470.8 g (target 245-585 grams).
- Housing: With the exception of the breeding period and dams with litters, animals were housed individually.
- Caging:
- Before the breeding, sires were housed singly in ventilated caging.
- Dams were housed singly in conventional shoebox caging prior to breeding and during the gestation period, then with their pups during the lactation period.
- During the breeding period, sire/dam pairings were housed in wire bottomed cages.
- During lactation, pups were housed with the dam in conventional shoebox caging.
- After weaning, pups were housed individually in ventilated caging until postnatal day 120, when they were transferred to shoebox caging due to their large size.
- Harlan TEK-Fresh diamond soft bedding replaced standard corn cob bedding during the gestation and lactation periods, and also when hematuria or diarrhea was observed, or other issues as specified by the veterinarian.
- All animals received plastic enrichment tubes only for environmental enrichment.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: I. Diet: 5K75 irradiated rat chow until arrival of custom diet, then
II. Purina AIN-93G diet – Irradiated from at least five days prior to breeding. This diet is formulated as a growth/lactation diet and was fed to all animals until postnatal day 95-99.
Samples of the diet were tested for aluminum, iron, manganese, copper, and zinc content.
Diet levels of aluminum were 6-9 ppm (6-9 µg/kg diet) over the study (Final Report/Draft, 2009, p.6).
- Water: deionized H2O, ad libitum.
Water levels of aluminium ranged from <1 – 160 ppb or 1 ug Al/L- 160 ug Al/L.
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26°C
- Humidity (%): 30 - 70%
- Air changes (per hr): ≥ 10 per hour in the roomand within ventilated cages, animals were expected to experience approximately 50 air changes per hour using room air for both intake and exhaust.
- Photoperiod (hrs dark / hrs light): ~12 hr. light

ADDITIONAL INFORMATION
- Number of Animals in the Study: The study began with 180 pairs to ensure 100 litters of progeny were delivered within the allotted timeframe (5 consecutive days);
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required mass of dry aluminium citrate was added to about 75% of the necessary volume of boiling deionised water on a hot plate (with stirrer). The mixture was then covered and heated to 96ºC until all the aluminium citrate was dissolved. After allowing the mixture to cool to room temperature, the pH was measured and adjusted to between 6 and 7 using sodium hydroxide and hydrochloric acid. The volume was then brought to a known value using deionised water to produce a “stock solution”. The stock solution was then filtered (0.45 µm) and stored in an interim vessel. Formulations were prepared weekly and stored in a plastic carboy at ambient temperature.

To produce the dosing solutions, a calculated volume of the filtered stock solution was measured into a carboy and diluted by the required amount with deionised water. The pH of the final dosing solution was measured to ensure that it was in the required range of 6 to 7.

Dosing solutions were transported to the animal test facility in 18L plastic carboys.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification of Al concentrations in the formulations and dosing solutions
The formulations and dosing solutions were prepared based on the Al content specified in the supplier’s Certificate of Analysis. Samples of at least 5 mL of each dose level of the dosing solution and also for the sodium citrate reference solution were stored and transported (overnight; ambient temperatures) then analyzed for aluminium content by ICPMS. Samples were collected from the first formulation, then from each week’s formulation for 4 weeks, then at 4 week intervals and, at the last dose preparation, until the end of the study.

The analyses showed that the dosing solutions prepared from the third lot of Al citrate had unexpectedly low Al concentrations, about 25% below target. The amount of Al citrate was thus increased to compensate. The Certificate of Analysis from the supplier gave a nominal concentration of 8.7% Al for this lot of the test item. The lower than specified Al levels (6.6% by analysis) were later confirmed by the supplier.

The Al concentrations in the dosing solutions differed from target by -30% to +39% throughout the study (Appendix B, Table B26. Dose Verification).

The stability and homogeneity of the dosing solutions under test conditions were determined in a separate study (Appendix F: Evaluation of the Stability and Homogeneity of Aluminium Citrate in Sodium Citrate and Aluminium Citrate Dosing Solutions by ICP-MS; ALB206-070144-5120). The results indicated that aluminium concentrations (at 2.5 g/L Al-citrate or endogenous Al levels in 27.2 g/L sodium citrate) remained stable and well-mixed in aqueous solution in a feeding bottle at room temperature for a 21 day period.

Aluminium Levels in the Diet and Vehicle
Samples of the different diets were analysed for aluminium, iron, manganese, copper, and zinc. For the enriched Purina AIN-93G, one sample was collected prior to the study and another was collected 6 weeks after the experimental starting date. One sample of Purina AIN-93M was taken prior to the switch in diets and another 6 weeks later. When new lots of the maintenance diet were received, they were tested before entering the study and again 6 weeks after being introduced.

Levels of aluminium in the diets were 6-9 ppm (6-9 µg/kg diet) over the study.

Levels of aluminium in the Nanopure water ranged from <1 – 160 ppb or 1 ug Al/L- 160 ug Al/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; see Appendix B, Table B26).

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.)
Remarks:
Doses / Concentrations:

Basis:
other: Low dose group (Group A): 30 mg Al/kg bw Mid dose group (Group D): 100 mg Al /kg bw High dose group (Group E): 300 mg Al/kg bw Control I: Distilled water Control II: Na citrate group (Group B): 27.2 g/L citrate
No. of animals per sex per dose:
Dams: 20/group;
Offspring: 10-20 females and 10-20 males/group;
Litters: 20 litter/dose.
Control animals:
other: A control group received water only. A citrate control group received sodium citrate at dose equimolar to citrate in the aluminium High dose group/300 mg Al/kg/Group E - 27.2 g/L.
Details on study design:
Dose Selection
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. After insemination, each dam was returned to her original cage and remained at that location until postnatal day 1 or euthanasia.

As the proportion of dams in each treatment groups that would deliver on a specific day could not be predicted, extra breeding pairs were included in the study. After the end of the week during which deliveries were expected, litters that were eligible to enter the study (≥4 pups of each sex) were randomly chosen to provide a balanced distribution of litters per treatment group per delivery day.

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 clinical examinations performed and frequency:
Parental animals
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:
- footsplay 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.
Specific biochemical examinations:
No specific neurobiochemical testing was performed.
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
Pups
Functional Observational Battery (FOB)
The Functional Observational Batteries (FOB) testing was conducted according to test facility Standard Operational Procedures (SOPs).

One male and one female pup from each litter in the Day 364 cohort (pups 4 and 8; see table above) underwent FOB examinations on PNDs 5 and 11 (during the neonatal period), on PND 22 (as juvenile pups), and as adults on PNDs 36, 45, 56, and then biweekly until the week of PND 350).

The tests on adults consisted of a cage-side assessment, a handling assessment, open field assessments and neuromuscular measurements (foot splay and fore- and hind-limb grip strength). The younger animals received abbreviated versions of the adult test. Observers were blinded to the treatment but whether the same technicians were used for all observations was not specified. The scales used for reporting the results are provided in the report.

The animals were tested in rack order as they had already been randomized to rack placement. Ten groups were tested (5 treatments and 2 sexes). To minimize any effect of time of day on the test results, the order of the animals from the 10 groups was randomized.
In addition, the animals from the Day 23, Day 64 and Day 120 cohorts received a 30 second open field assessment within the two days before sacrifice to capture any “overtly abnormal behaviour”.


LOCOMOTOR ACTIVITY: Yes
Motor Activity
Motor Activity testing was conducted on all animals from the Day 23, Day 64, Day 120 and Day 364 cohorts according to a test facility SOP.

Motor activity was monitored on PNDs 15 or 16, 17 and 21 in the Day 23 cohort animals, on PND 62 in the Day 64 cohort animals, on PND 117 in the Day 120 cohort animals, and on PND 363 in the Day 364 cohort animals. Animals in the Day 23 cohort were tested three times to assess the development of motor activity while, to avoid effects from habituation, the animals in the other cohorts were tested only once. The same animals were evaluated at each pre-weaning time-point in the Day 23 cohort.

A 24 station Photobeam Activity System-Home Cage (PAS-HC) with a 4 x 8 photobeam configuration (San Diego Instruments , Inc., San Diego, California, USA), was employed with standard transparent plastic cages.

Twenty-four individual testing chambers were used and tested animals were assigned to chambers using a Youden Square to minimize possible spatial bias. The treatment groups were also counterbalanced for the multiple testing devices.

Animals were tested for one 60-minute session, which was divided into 12 five minute intervals for reporting. An exception was the Day 64 cohortfor which the session was mistakenly divided into 300 twelve-second intervals. The resulting data were summed for each set of 25 twelve-second intervals to give intervals of 5 minutes for reporting. Given the nature of the test, although a deviation from protocol, this is unlikely to have had an important effect on the results.

Auditory Startle Response
Auditory startle response was monitored on postnatal days 21, 62, 117 and 363 and was conducted according to a test facility SOP. The instrumentation used was an 8-station SR-LAB™ Startle Response System (San Diego Instruments Inc, San Diego, California, USA).

As per the testing schedule, all animals in the four cohorts underwent Auditory Startle testing before sacrifice. The day of testing was counterbalanced across treated and control groups. The test facility had 8 testing chambers. Therefore, the treatment groups were also counterbalanced for the use of multiple testing devices. All test subjects were assigned to chambers using a Youden Square to minimize possible spatial bias.

Each trial consisted of a startle stimulus (120dbB) of 50-ms duration with 2-ms rise/fall time. There were 5 blocks of 10 trials per session and the mean response amplitude on each block of 10 trials was recorded. Animals were given a 5 minute acclimation period in the test chamber with a 65 db background level before each test session.



AUDITORY STARTLE REFLEX HABITUATION: No data


LEARNING AND MEMORY TESTING: Yes
(T-maze)
The T-maze test of memory and spatial recognition was conducted according to a test facility SOP on one female and one male weanling pup per litter in the Day 23 cohort (PND 22). The specific test used was a spontaneous alternation test with no reinforcement. This test assesses an animal's inclination to explore new areas.

The T-maze used in the tests was from San Diego Instruments (San Diego, California, USA) or one made to the same specifications. Visual cues, for example stripes and geometrical shapes, were on the inside walls of the T-maze to help guide the rats.

The study consisted of two 5 minute trials separated by four to six hours. In the first trial, with one arm of the maze blocked (right or left counterbalanced across treatment groups), the animals were allowed to explore for five minutes. In the second trial, the animals explored the maze with both arms unblocked. A computer program (T-maze, Dr. Joseph Rochford, University of Montreal) was used to record the number of visits to each arm of the T maze and the time interval of each visit during each 5 minute trial. An animal was considered to have entered the other arm if an imaginary line between its front toes crossed the boundary. “Alternation” had occurred if an animal entered the previously blocked arm during the second trial. This definition of “alternation” differs from that in the original study plan. However, the authors considered the new definition more consistent with the literature and simpler to measure and interpret.

Animals were tested in rack order with counterbalancing for the time of day of testing across treatment groups. All tests were conducted during the light period.

The Morris Water Maze tests
The Morris Water Maze test was conducted according to test facility SOPs prior to PND 59, 61, 63 and 64 (Day 64 cohort animals), prior to PND 114, 116, 118 and 119 (Day 120 cohort animals), and prior to PND 357, 359, 361 and 362 (Day 364 cohort animals).

The study consisted of three blocks of four trials for a total of 12 training trials. These trials were conducted over 3 alternate testing days with one day for each block. A block of probe trials was conducted on the day after the last of the three blocks.

The tests were carried out in a 1.4 m diameter black plastic tub with four quadrants filled to about 40-50 cm depth with water made opaque with black tempra paint. A 10 cm square submerged (invisible) platform (San Diego Instruments, San Diego, California, USA) was in the target quadrant.

Training trials: For training, the animals were introduced into one of three of the non-target quadrants in the tub. Spatial cues were provided to the animals by large shapes on the walls in the testing room. The time taken for the animals to find the platform was recorded for each run.

Platform-removed probe trials: Platform-removed probe trials consisted of releasing the animals from a non-target quadrant, tracing their paths on a form and recording the time spent in each quadrant (using a computer program, Quadrant.exe), for a period of one minute.

The release points were changed in a random sequence in the Day 64 and Day 129 cohorts. In the Day 364 cohort trials, the same release point was used for all the animals in each trial for a given day with randomization between trials.

For each trial, the following were recorded to describe the search strategy employed: the sequence of quadrants entered: the time spent in each quadrant (especially the target quadrant), and whether the rats swam directly or indirectly to the target quadrant. The time to reach the platform was also recorded (latency).

Visible platform probe trials: In these trials, the water level was dropped slightly so that the platform was visible to the animals on release. The latency (time to reach platform) was measured and traces of the swim path recorded.

Thigmotaxis (circling behaviour close to the periphery of the pool indicative of anxiety) was analyzed visually as “wall-hugging“. The swim path trace for each trial was examined for wall-hugging.

Animals were tested in rack order with counterbalancing for the time of day of testing across treatment groups. All tests were conducted during the light period.
Sacrifice and (histo)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.

At the discretion of the study director, animals from the perfusion group found dead or euthanized prior to the end of the study underwent a regular necropsy conducted by or under the supervision of a veterinarian.

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”.

Examples of particular changes of interest were provided:
1. Gross changes in the size or shape of brain regions such as alterations in the size of the cerebral hemispheres or the normal pattern of foliation of the cerebellum.
2. The death of neuronal precursors, abnormal proliferation, or abnormal migration as indicated by pyknotic cells, or ectopic neurons, or gross alterations in regions with active proliferative and migratory zones, alterations in transient developmental structures (e.g., the external germinal zone of the cerebellum).
3. Abnormal differentiation, while more apparent with special stains, may also be indicated by shrunken and malformed cell bodies.
4. Evidence of hydrocephalus, in particular enlargement of the ventricles, stenosis of the cerebral aqueduct and general thinning of the cerebral hemispheres.

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 methylmethacrylate (GMA), sectioned and stained with hematoxylin 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:
None.
Positive control:
No positive control group exhibiting central nervous system pathology and peripheral nervous system pathology available.
Statistics:
For details see "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
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, treatment-related
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):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.

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.


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.

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.

(migrated information)
Details on results:
Mortality
Based on Appendix B, Table B1 (Necropsy disposition check), 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 (#4695; Appendix B, Table B3; injuries from a fight).
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.

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.





Dose descriptor:
NOAEL
Effect level:
3 225 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Maternal Toxicity, rat, body weight
Remarks on result:
other: Generation: maternal (migrated information)
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
element
Remarks:
Al 3+
Sex:
female
Basis for effect level:
other: Maternal Toxicity, rat, body weight
Remarks on result:
other: Generation: maternal (migrated information)
Dose descriptor:
NOAEL
Effect level:
ca. 1 907 mg/kg bw/day
Based on:
other: For Aluminium sulphate (convert using 342.1 g/mol)
Sex:
female
Basis for effect level:
other: Maternal Toxicity, rat, body weight
Remarks on result:
other: Generation: maternal (migrated information)
Dose descriptor:
LOAEL
Effect level:
1 075 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Neuromuscular effects, hindlimb grip strength, forelimb grip strength
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day
Based on:
element
Remarks:
Al 3+
Basis for effect level:
other: Neuromuscular effects, hindlimb grip strength, forelimb grip strength
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
LOAEL
Effect level:
ca. 634 mg/kg bw/day
Based on:
other: Aluminium sulphate
Basis for effect level:
other: Neuromuscular effects
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
NOAEL
Effect level:
323 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Neuromuscular effects;
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
element
Remarks:
Al 3+
Basis for effect level:
other: Neuromuscular effects;
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
NOAEL
Effect level:
ca. 190 mg/kg bw/day
Based on:
other: For Aluminium sulphate using 342.1 g/mol
Basis for effect level:
other: Neuromuscular effects
Remarks on result:
other: Generation: offspring (migrated information)

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:

Parameter

Pairwise Differences

Absolute Agranulocytes

Levels in the high dose group were significantly less than in the control group (p=0.0198).

Absolute Granulocytes

Not significant (ns)

Agranulocytes

Ns

Granulocytes

Ns

HCT (haematocrit)

The high dose group had significantly lower HCT than all the other groups (control group, p=0.0016; low dose group, p=0.0019; mid-dose group, p=0.0067; Na-citrate, p=0.024).

HGB (haemoglobin)

The high dose group had significantly lower HGB than in the controls (p=0.0034), the low dose group (p=0.0089), and the mid-dose group (p=0.0027).

MCH (mean cell haemoglobin)

The high dose group had significantly lower MCH than all the other groups.

MCHC (mean cell haemoglobin concentration)

The mid-dose group had significantly higher levels than the low-dose and Na-citrate groups.

MCV (mean cell volume)

The high dose group had significantly lower MCV than all of the other groups.

 

PLT (platelet count)

Ns

NUC_RBC (nucleated red blood cells)

Zero

RBC (red blood cell count)

The red blood cell count in the Na-citrate group was significantly less than in the high dose group.

 

WBC (white blood cell count)

The high dose group had significantly higher numbers of WBC than the control and low dose groups.

 

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.

 

 

 

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

The results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. An effect of Na-citrate was observed in the female pups. Urinary tract pathology was observed in high dose rats, more frequently in the males. The results showed no evidence of an effect on memory or learning. Fairly consistent results were observed for the critical effect, fore- and hind-limb grip strength, and this was supported by the following less consistently observed effects also observed in the mid-dose (100 mg Al/kg bw/day) group: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools that controls); tail pinch (mid-dose females displayed more exaggerated responses); foot splay (mid-dose females had significantly narrower foot splay than the controls); the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls). No treatment-related differences in FOB characteristics were observed in the neonatal and juvenile pups. A LOAEL of 100 mg Al/kg bw/day for aluminium repeated dose toxicity is assigned based on this study.
Delayed sexual maturation, measured as delayed vaginal opening in females and delayed preputial separation in males, was observed in the high dose Al-citrate group of this study. The same effect, although somewhat less pronounced, was also seen in the sodium citrate control group. Based on the observed upward deviations from the target dose in the Al citrate groups and the data on water consumption seen in the first weeks after weaning, it is possible that both in the pre- and post-weaning stage, the animals in the Al citrate groups received considerably more citrate than the sodium citrate control group. Moreover, the calculated Al dose during the immediate post-weaning period was more than twice the target dose, which may have contributed to post-natal systemic toxicity due to exposure to the test substance. Given that effects were seen in both the Al-citrate high-dose group and the NA-citrate group, no Al-based LOAEL/NOAEL can be suggested based on the sexual maturation results in this study.

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

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 (nominal - 30, 100 and 300 mg Al/kg bw/day. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.

 

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

 

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

 

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

 

The most notable treatment-related effect observed in the offspring was renal pathology – hydronephrosis, ureteral dilation, obstruction and presence of calculi - most prominently in the male pups. Higher mortality and significant morbidity were observed in the male pups in the high dose group; leading to euthanization of this group atca. study day 89. Clinical observations that showed a relationship with treatment, either directly or secondary to renal failure, were poor coat, weight loss, and haematuria. Diarrhoea was also observed. These signs were found only in the high dose Al-citrate treatment group. Haematuria was also observed in some animals in the Na-citrate group in the Day 364 cohort. Dosing with Al-citrate was associated with a reduction in body weight. The results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. In the Day 120 cohort male pups, the mid-dose animals were significantly lighter than the controls. An effect of Na-citrate was observed in the female pups in the Day 364 cohort. Overall, dosing of animals with aluminium citrate led to higher fluid consumption than in the control animals. Dosing with Na-citrate was associated with a significant increase in fluid consumption relative to that of the controls in most cohorts, with the exception of the Day 64 cohort females (fluid consumption was significantly lower in the Na-citrate group) and the Day 364 males (no significant difference between the two groups). The animals’ fluid consumption varied with time and, in mature animals, was less than expected (120 mL/kg bw/day) with implications for the actual dosage of test item received. Despite the deviations from the target dose, the low-, mid- and high-dose groups showed the required trend of lowest to highest maintaining statistically significant group differences in dose levels. For most of the study period, the actual dose received was less than the target dose in all treatment groups.

 

In the female pups, the mean number of days to reach vaginal opening was 31.3 (±2.1, sd) in the control group and 39.7 (±5.6, sd) in the high dose Al-citrate group, a significant difference (p<0.0001). In males, the mean number of days to reach preputial separation was 39.6 (±2.1, sd) in the control group and 42.5 (±3.2, sd) in the high dose group, also a significant difference in the pair-wise comparisons (p<0.0001). Delayed development of both male and female pups was observed in the high dose Al-citrate and Na-citrate groups. The effect is considered treatment-related but whether the effect is secondary to decreases in body weight is not clear, however.

 

FOB observations showed no clear treatment-related effect among the neonatal Day 364 cohort pups that were assessed at PND 5 and 11 or in the juvenile pups assessed ca.PND 22. In the adult pups, the data provide little evidence for an Al effect on the autonomic function domain, the sensimotor function domain, or excitability. Significant wasting (physiological domain), was observed in the high dose females and appears related to treatment. Characteristics of defecation (number of boluses) also showed differences with treatment. In addition, there was limited evidence of effects on activity/well-being of the pups at the high dose as reflected in fur appearance, deposits and rearing. There was some evidence for dose-response relationships between neuromuscular measurements – hind-limb and fore-limb grip strength and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. Although the FOB endpoint most consistently associated with Al-citrate treatment, grip strength, measurements showed considerably variability and a consistent ordering of the Al-treatment group responses (dose-response) was not observed at all time points. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning Day 23 cohort) or the Morris Water Maze test (Day 120 cohort).

 

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

 

Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control groups. There were no significant sex differences. These results suggest transfer of Al from dams to pupsin utero, although a contribution from breast milk PND 0 to 4 is also possible. Aluminium levels were assayed in several tissues in the pup cohorts. Levels of Al in whole blood were highest in the Day 23 cohort animals and declined with time, possibly due to the lower amounts of water (test solution) consumed once the pups matured. Although during the lactation period pups may have consumed some water/test solution, the results suggest that transfer of Al from dams to pups can occur through breast milk. 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 much strength. 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 MTD. 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. The report mentions that the observation protocol was not always consistently followed and that the clinical observations for the Group B and E animals started on April 3, 2008 instead of March 19, 2008. Some auditory startle data was lost due to an equipment malfunction (16 animals in Cohort 1 and 7 animals in Cohort 2) resulting in a “loss of statistical power for this part of the study”. Some necropsies were not completed “22 pre-weaning animals (eight from one litter) and 3 post-weaning animals that died or were euthanized prior to scheduled sacrifice did not have a necropsy completed”. The equipment used to measure grip strength had a maximum capacity of 700g leading to the possibility of underestimation of grip strength in larger animals, particularly in the males. Overall, these deviations were unlikely to have impacted the results of the study.

The results from this study are informative for developmental and 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).As the offspring were dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. Urinary tract pathology was observed in rats in the high dose group, more frequently and more severe in the males. The study showed no evidence of an effect of Al-citrate on memory or learning but a more consistent effect was observed in endpoints in the neuromuscular domain.

 

The ambiguity as to the critical period of exposure and the time-varying water consumption complicate the derivation of a point-of-departure from this study. A LOAEL of 100 mg Al/kg bw/day for aluminium toxicity is assigned. The critical effect was a deficit in fore- and hind-limb grip strength in the mid-dose group, supported by evidence of dose response and less consistently observed effects in the mid-dose animals: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools than controls); tail pinch (mid-dose females displayed more exaggerated responses); foot-splay (mid-dose females had significantly narrower foot-splay than the controls); and the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
190 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information as a whole meets the tonnage driven data requirement of REACH. Moreover, reliability and consistency are observed across the different studies (see discussion below).

Effect on neurotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There were no studies available in which the neurotoxic properties of aluminium sulfate were investigated.Information available on aluminium compounds were therefore considered for this endpoint, since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+).

Therefore, a recent combined one-year developmental and chronic neurotoxicity study with Al-citrate was conducted to determine whether aluminium administered at near toxic concetrations in its salt form, could significantly affect brain physiology and compromise higher function such as memory and motor activity (Poirier et al, 2011,Alberta Research Council Inc, 2010)

This study is of interest for the evaluation of the neurotoxicity of aluminium sulfate, taking into consideration the bioavailability of aluminium sulfate compared to Al-citrate (see toxicokinetic section) 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.

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.

Finally, a conservative NOAEL of 322 mg Al-citrate/kg bw/d corresponding to 30 mg Al/kg bw/d was derived from this study. This would correspond to ca. 190 mg/ kg bw/d of aluminium sulfate as anhydrous form (molecular mass of 342,1 g/mol).

Concern was raised about Aluminium exposure and neurodegenerative diseases while numerous epidemiological studies have been carried out to try to determine the validity of this hypothesis. These have been reviewed in detail by several authorities (JECFA, 2012, COT, 2005, ATSDR, 2008, ECH, 2010). Investigators have identified a number of difficulties in carrying out such studies on conditions for which the causes are multifactorial. In addition, there are questions regarding the levels of exposure to aluminium from different sources and the relative bioavailability from these sources. Most of the studies have focused on aluminium in drinking water—although this is a very minor source of exposure—and Alzheimer disease. Most of the studies do not consider the speciation of aluminium, and the assessment of exposure from both drinking-water and food is usually not well characterized. In particular, there are difficulties in determining recollected exposure when the subject has a degenerative neural condition affecting cognitive performance. The conclusion of the recent JECFA evaluation (2012) was that “some of the epidemiology studies suggest the possibility of an association of Alzheimer disease with aluminium in water, but other studies do not confirm this association.

All studies lack information on ingestion of aluminium from food and how concentrations of aluminium in food affect the association between aluminium in water and Alzheimer disease.” There are suggestions that persons with some genetic variants may absorb more aluminium than others, but there is a need for more analytical research to determine whether aluminium from various sources has a significant causal association with Alzheimer disease and other neurodegenerative diseases (WHO, 2013).

Both EFSA (2008,2001) and JECFA (2012) concluded that the information available was inconsistent and did not support a causal association between aluminium exposure and Alzheimer’s disease or other chronic neurological diseases. SCCS (2014) considers that Aluminium (Al) is a known neurotoxicant and circumstantial evidence has linked this metal with several neurodegenerative disorders like Alzheimer's disease, Parkinson’s diseases and other chronic neurodegenerative diseases, but no causal relationship has yet been proven.

In agreement with the general scientific concensus, the available data on neurotoxicity provides sufficient evidence that Aluminium sulphate is not neurotoxic to animals or to humans.


Justification for selection of effect on neurotoxicity via oral route endpoint:
No guideline Study was available on Aluminum sulphate. Read- across approach using the key study performed on Aluminum citrate and conducted according to OECD guideline 426 in compliance with GLP is considered as adequate to assess the neurotoxicity of aluminium sulfate (see §"Toxicokinetics").

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available for neurotoxicity by oral and dermal route , by inhalation for Aluminium sulphate

Self classification:

Based on available data, Aluminium sulphate is not classified for neurotoxicity according to the Regulation (EC) No. 1272/2008 and the Directive 67/548/EEC criteria.