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Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
11 September 2006 - 03 November 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD guideline 422 and under GLP conditions.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation:
Males: approx. 9 weeks
Females: approx. 11 weeks
- Weight at study initiation:
Males: mean 266-269 g, SD 6.6-12.2
Females: mean 237-240, SD 7.0-10.7
- Housing: According to guideline
Pre-mating: groups of 5 animals/sex/cage
Mating: 1 male, 1 female
Post-mating: males in groups of 5 animals/sex/cage, females individually
Lactation: offspring together with dam
- Diet (e.g. ad libitum): Ad libitum, pelleted rodent diet
- Water (e.g. ad libitum): Ad libitum, tap-water
- Acclimation period: 4 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-23.4
- Humidity (%): 37-95 (guideline: max. 70%, but no effects expected)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not relevant
Vehicle:
other: Water (Milli-U)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance. No adjustment was made for specific gravity of the vehicle and formulation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: ?
- Amount of vehicle (if gavage): 5 ml/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: Not relevant
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulated samples were analysed using ICP-MS
Duration of treatment / exposure:
Males: 28 days (14 days prior to and 14 days during mating)
Females: 28 days (14 days prior to and 14 days during mating) + 9-25 days (during gestation and lactation)
Frequency of treatment:
Once daily, 7 days a week
Details on study schedule:
Not relevant
Remarks:
Doses / Concentrations:
40, 200, 1000 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
3.6,18,90 mg Al 3+/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on the results of a dose range finding study (NOTOX project 473524)
- Rationale for animal assignment (if not random): Random
- Section schedule rationale (if not random): No data
Positive control:
Not relevant
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations checked: Mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: At study start, once weekly and at death (females: gestation days 0, 4,7, 11, 17, 20, lactation day 1, 4)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

WATER CONSUMPTION: Subjective appraisal but not recorded

OTHER:
FUNCTIONAL OBSERVATIONS:
Males during week 4, females during lactation; 5 M and5F, randomly selected from all groups:
Hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity test (recorded 12 hrs overnight)

REPRODUCTIVE BEHAVIOUR:
Male number paired with, mating data, confirmation of pregnancy, delivery day

Oestrous cyclicity (parental animals):
Not performed
Sperm parameters (parental animals):
No data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Not applicable

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
numbers of live and dead pups( daily)
individual bw on day 1 and 4 of lactation
sex of all pups on day 1 and day 4 by assessment of ano-genital distance
physical or behavioural abnormalities daily

GROSS EXAMINATION OF DEAD PUPS:
Yes, sexed and externally examined if practically possible. The stomach was examined for the presence of milk.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on day 29 of study
- Maternal animals: All surviving animals on day 4 post partum

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination of all organs of all animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] (see "Any other information on materials and methods incl. tables) were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
The F1 offspring was sacrificed at 4 days of age.

GROSS NECROPSY
All offspring sexed and externally examined. Stomach examined for the presence of milk
All macroscopic abnormalities recorded, and defects or cause of death evaluated. Any abnormal pup preserved in 10% buffered formaldehyde
Statistics:
Dunnet -Test (Dunnet, 1955) based on pooled variable estimate for variables assumed to have a normal distribution
Sett-test (Miller, 1981) for variables asssumed not to follow a normal distribution
Student’s T- test for pup organ weights
Fisher exact test for frequency data. All tests two-sided, P<0.05
Reproductive indices:
Percentage mating
Fertility index
Conception rate
Gestation index
Offspring viability indices:
Percentage live male & female pups
Percentage post-natal loss
Viability index
Clinical signs:
no effects observed
Description (incidence and severity):
Observations: Mortality / viability at least twice daily Detailed clinical signs at least once daily Body weights at start, once weekly and at death (females: gestation days 0, 4,7, 11, 17, 20, lactation day 1, 4 food consumtion weekly water consumption
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See RS on 28-day repeated dose part of study
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See RS on 28-day repeated dose part of study
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ body weights: From 5 surviving animals/sex/group*: Adrenal glands Liver Brain Spleen Epididymides Testes Heart Thymus Kidneys
Gross pathological findings:
no effects observed
Description (incidence and severity):
Parental animals: Macroscopic examination of all organs of all animals Microscopic examination of following organs from 5 animals/sex/group: Identification marks: not processed Ovarie ovaries Adrenal glands Pancreas Aorta Peyer's patches (jejunum, ile
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histopatological abnormalities were obsurved
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Dose volume 5 ml/kg bw. 0, 40, 200, 1000 mg/kg/day of the test substance solution, equal to 0, 3.6, 18, 90 mg/kg bw/ d of Al3+
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
M/F ratio : 1/1 per cage Maximum of 14 days, separation once mating occurred (i.e evidence of sperm in vaginal lavage or intravaginal copulatory plug) Proof of pregnancy: littering and post-mortem examination of reproductive organs
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
% pregnant per dose level: see under details on results
For Food consumption , Clinical signs, Haematological findings and duration, Clinical Biochemistry findings, Gross pathology : See RS on 28-day repeated dose part of study


Body weight (parenteral animals)
Slightly lower mean bw for females at 1000 mg/kg bw/d (90 mg/kg bw/d) at day 8 with slight weight loss for three of these females. BW recovered during the following mating and post-coital period. In the other groups no deviations compared to controls

Parameters assessment during study (maternal and fetal)
Clinical signs as above
Percentage mating
Fertility index
Conception rate
Gestation index
Percentage live male & female pups
Percentage post-natal loss
Viability index

% pregnant per dose level:
Dose (mg/kg bw/d) 0 40 200 1000
% pregnant 100 90 90 90

Number aborting:
None

Number of resorptions, early/late if available:
Number of implantations:
Pre and post implantation loss, if available; Number of corpora lutea
Corpora lutea:
no significant differences between exposed and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Corpora lutea 18.5 (2.01) 18.2 (7.94) 15.0 (5.40) 16.9 (1.91)
Implantation sites:
no significant differences between exposed and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Inplantation sitea 16.6 (2.55) 15.7 (5.72) 14.0 (5.35); 16.2 (1.99)
Litter size:
no significant differences between exposed and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Litter size 15.1 (3.1) 14.0 (5.1) 14.7
(2.7) 14.8
(1.9)

Duration of Pregnancy:
No difference between dose groups and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
MF ratio 21.9 (0.3) 21.9
(0.6) 21.9
(0.3) 21.8
(0.4)





Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No abnormalities at any dose level
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
Aluminium (Al 3+)
Sex:
female
Basis for effect level:
other: No abnormalities at any dose level
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
No difference between exposed and controls Alive / dead (nrs per group): Dose (mg/kg bw/d) 0 40 200 1000 Live /dead 136/0 126/12 132/1 148/0
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Litter size: no significant differences between exposed and controls Mean (SD): Dose (mg/kg bw/d) 0 40 200 1000 Litter size 15.1 (3.1) 14.0 (5.1) 14.7 (2.7) 14.8 (1.9) Litter weights: No difference between exposed and controls Lactation day 1; M
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Grossly visible abnormalities, external, soft tissue and skeletal abnormalities : No abnormalities observed either in controls or in exposed animals ouside the normal biological variation for race/strain No behavioural abnormalities in pups observed
Histopathological findings:
not examined
Sex ratio
No difference between exposed and controls
M / F ratio:
Dose (mg/kg bw/d) 0 40 200 1000
MF ratio 1.1 0.84 0.99 0.92


Postnatal growth
No difference between exposed and controls
Postnatal weight, Lactation day 4; Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Weight (gr) 10.5 (1.5) 10.3 (0.9) 10.5 (1.4) 10.4 (0.6)

Postnatal survival
No difference between exposed and controls
Post-natal pup deaths:
Dose (mg/kg bw /d 0 40 200 1000
Nr. Of dead pups 1 2 0 3

Organs examined at necropsy (macroscopic and microscopic)
Pups:
All offspring sexed and externally examined. Stomach examined for the presence of milk
All macroscopic abnormalities recorded, and defects or cause of death evaluated. Any abnormal pup preserved in 10% buffered formaldehyde


Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No abnormalities at any dose level
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
Aluminium (Al 3+)
Sex:
male/female
Basis for effect level:
other: No abnormalities at any dose level
Reproductive effects observed:
not specified

1 female of the control group sacrified in extremis at day 22 of pregnancy due to parturition difficulties

Conclusions:
This study revealed no maternal toxicity at any dose and no reproductive, breeding or developmental toxicity at any dose from two weeks prior to mating to at least 3 days of lactation (females). Therefore, a NOAEL for maternal local and systemic toxicity of 1000 mg/kg bw/d (equivalent to 90 mg/kg/d Al3+) was established. For reproductive and developmental toxicity the NOAEL was 1000 mg/kg bw /d (equivalent to 90 mg/kg/d Al3+).
Executive summary:

A combined repeated dose / reproductive screening study (OECD 422), studied the administration of Aluminium chloride basic by oral gavage to male and female Wistar rats at dose levels of 40, 200 or 1000 mg /kg bw/day (equivalent to 3.6, 18 or 90 mg/kg bw/day Al3 +). 10 animals/sex/group were used. Males were exposed for 28 days, females between 37 -53 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 3 days of lactation. Litter was not exposed.

There were no treatment-related effects found regarding to mortality, clinical signs, functional observations, histopathology, organ weigths, reproduction, breeding data and pup development. No reproduction, breeding and developmental toxicity was observed for treatment up to 1000 mg/kg bw/day of aluminium chloride basic (equivalent to 90 mg/kg bw/day Al3+).

Based on these results, the No Observed Adverse Effect Level for maternal and developmental toxicity was established to be 1000 mg/kg bw/day for Aluminium chloride basic (equivalent to 90 mg/kg bw/day Al3+)

In a combined repeated dose / reproductive screening study, administration of Aluminium chloride basic by oral gavage to male and female rats at dose levels of 20, 200 or 1000 mg /kg bw/d,

From two weeks prior to mating to at least 3 days of lactation (females) or 28 days (males) revealed no maternal toxicity at any dose and no reproductive, breeding or developmental toxicity at any dose.

The NOAEL for paternal systemic toxicity is 1000 mg/kg bw/d (equivalent to 90 mg/kg/d Al3+).

The NOAEL for maternal local and systemic toxicity is 1000 mg/kg bw/d (equivalent to 90 mg/kg/d Al3+).

The NOAEL for reproductive and developental toxicity is 1000 mg/kg bw /d (equivalent to 90 mg/kg/d Al3+)
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 142 mg/kg bw/day
Study duration:
subacute
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 fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In terms of hazard assessment of toxic effects, available data on the toxicity to reproduction/development of other aluminium compounds was taken into account by read-across following a structural analogue approach, since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+) as described in the toxicokinetics section.

There are 2 two-generation studies available to support a hazard assessment of the reproductive effects of aluminium. However, interpretation of both studies for risk assessment is limited since effects reported may be due to limited water consumption observed in the study. Currently, two GLP studies on reproductive/developmental toxicity are available, one with aluminium sulfate and the other with aluminium ammonium sulfate which is relevant for the assessement as similar aluminium soluble salt. These studies are described below.

In the two OECD TG 416 and GLP compliant studies, aluminium sulfate Al2(SO4)3 (AS) and aluminium ammonium sulfate (AAS) (CAS#: 7784-25-0 as anhydrous)) CAS#: 7784-26-1 as dodecahydrate] were administered by a relevant oral route with drinking water to Crl: CD(SD) rats at multiple dose levels (120, 600 and 3000 ppm and at 0, 50, 500 or 5000 ppm, respectively)(Hirata-Koizumi et al., 2011a & b). Twenty-four animals per sex and group (F0 and F1 generation) were given AS and AAS in pH 3.57 - 4.20 drinking water beginning at 5 weeks of age for 10 weeks until mating, during mating, throughout gestation and lactation. Litters were normalized on PND 4. In the F1 generation, 24 male and 24 female weanlings were identified as parents on PNDs 21 to 25, ensuring an equal distribution of body weights across groups. Drinking water provided to the F1 offspring contained the identical AS/AAS concentrations as those of their parents. These animals were then mated, and followed through gestation and lactation until sacrifice on PND 26. Each female was mated with a single male receiving the same AS/AAS drinking water concentration; if successful mating did not occur (as evidenced by sperm in a vaginal smear or presence of a vaginal plug) within the two week mating period, then the female was put in with another male from the same group who had mated successfully.

 Observations assessed in the parental animals included clinical signs of toxicity, estrous cycle, copulation, fertility, gestation (including numbers of implantations) and delivery indices, the numbers of testis and cauda epididymal sperm, sperm swimming speed, percentage of motile sperm, percentages of motile sperm and percentages of morphologically abnormal sperm. Litter parameters recorded at parturition (post-natal day zero; PND0) included the number of live and dead offspring and the numbers and types of gross malformations. Developmental landmarks assessed in the F1 and F2 pups were: body weight (daily); sex ratios, pinna unfolding PND1 to PND4; anogenital distance on PND 4; incisor eruption (in one male and one female pup per dam) beginning on PND 8; eye opening beginning on PND 12; surface righting reflex (PND 5), negative geotaxis (PND 8); and mid-air righting reflex (PND 18) in one male and one female pup per litter. In the F1 pups selected as F1 parents, the males were observed for timing of preputial separation (starting on PND 35) and the females were observed for timing of vaginal opening (starting on PND 25). Neurobehavioral testing was conducted at two time points in randomly selected offspring (locomotor activity and T maze test).

The major findings in the aluminium sulfate study (Hirata-Koizumi et al., 2011a) include decreased drinking water consumption for both sexes in all AS groups, variable reductions in food consumption, reduced body weight in pre-weaning animals at 3000 ppm, delayed sexual maturation of the female F1 offspring at 3000 ppm, and decreased absolute liver, epididymides, thymus and spleen weight in the offspring at 3000 ppm. The authors proposed a LOAEL for aluminium sulfate for parental systemic toxicity and reproductive developmental toxicity of 31.2 mg Al/kg bw/day (3000 ppm) and NOAEL at 8.06 mg Al/kg bw/day (600 ppm). However, the authors state, correctly, that because “paired-comparison data are not available to assess the effects of decreased water intake in the absence of AS exposure” there is a possibility that the decreased absolute organ weights as well as delayed vaginal opening in the F1 females is likely secondary to the reduced body weight. The statistically significant delay in F1 female vaginal opening (29.5 ± 2.1 in controls and 31.4 ± 1.7 days in the highest dose group) was also not accompanied by adverse changes in estrous cyclicity, anogenital distance or further reproductive performance. It is likely that the observed effects are secondary to the reduced body weight development.The reduction in bodyweight is in turn likely to be related to the reduced food and water intake and a substance specific effect cannot be deduced from this study. Moreover "a clear impact on the hormonal event" was found to be unlikely by the authors as AS levels added to drinking water were 190, 946 and 4700 times greater than Al levels found naturally in drinking water (ca. 0.1 mg/L). In conclusion the authors suggested their NOAEL wa conservative.

The results presented on AAS (Hirata-Koizumi et al. 2011b) provide no evidence that prolonged consumption of AAS has an adverse impact on copulation, fertility and reproductive success in male and female Crl: CD(SD) rats consuming up to 517 mg AAS/kg-day. In discussing their data, the authors concluded that “copulation, fertility or gestation indices were not affected up to the highest dose tested at which average Al intake from food and drinking water was estimated to be 36.3 - 61.1 mg Al/kg per day. ”

The authors identified a LOAEL of 5000 mg AAS/L for both parental toxicity and reproductive toxicity (based on reduced pre-weaning body weight gain in F1 male (at PND 21) and female (PND 14, 21) pups, delay in the vaginal opening in F1 female pups, potentially attributed to inhibition of growth and decreased organ weights in F1 and F2 male and female offspring). The suggested LOAEL level corresponds to 36.3 mg Al/kg bw per day. The reported NOAEL is 500 mg AAS/L which corresponds to 5.35 mg Al/kg bw per day.

It has to be pointed out, that the interpretation of the results of both studies is difficult due to the clear effect of AS/AAS treatment on fluid consumption. Addition of AS to drinking water at high concentrations led to reduced pH (3.57 to 4.2) and this appears to have reduced the palatability of the drinking water. At these AS/AAS levels, the F0 and F1 females also decreased their food consumption relative to the controls. As a result, the reported observations represent secondary effects due to maternal dehydration and reduced nursing that may have influenced pup weight on PND 21 due to decreased drinking water consumption and decreased food consumption of F0 and F1 dams during the later stages of lactation. Consequently, the utility of this study for risk assessment is limited since the noted effects could be related more to decreased maternal fluid consumption than caused directly by the ingested substance.

In addition, a recent combined one-year developmental and chronic neurotoxicity study with Al-citrate was conducted to determine whether aluminium administered at near toxic concentrations 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 (20 per group) were administered aqueous solutions 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) and 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day) via drinking water, respectively. 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+. However, in a conservative approach, the NOAEL was reported based on this effect as 30 mg Al/kg bw/d.

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 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 for neuromuscular effects from this study. This would correspond to ca. 187 mg Al from aluminium sulfate as anhydrous form (molecular mass of 337,17 g/mol.

Moreover, the combined repeated dose / reproductive screening study (OECD 422) of Beekhuijzen (2007), studied the administration of Aluminium chloride basic by oral gavage to male and female Wistar rats (10 animals/sex/group) at dose levels of 40, 200 or 1000 mg /kg bw/d(equivalent to 3.6, 18 or 90 mg Al3+/kg bw/day). Males were exposed for 28 days, females between 37 -53 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 3 days of lactation. Litter was not exposed.There were no treatment-related effects found regarding to mortality, clinical signs, functional observations, histopathology, organ weigths, reproduction, breeding data and pup development. No reproduction, breeding and developmental toxicity was observed for treatment up to 1000 mg/kg bw/day.Based on these results, the No Observed Adverse Effect Level for maternal and developmental toxicity including the fertility was established to be 1000 mg/kg bw/day for Aluminium chloride basic which corresponded to 90 mg Al/kg bw/day.This study conducted on Aluminium chloride basic is relevant for Aluminium sulfate. Indeed, following ingestion Aluminium chloride basic is dissociated into the aluminium (Al3+) and the chloride (Cl-) in the acidic aqueous conditions of the gut such as the stomach.Therefore, as for Aluminium sulfate (see § 7.1), the toxicity of Aluminium chloride basic should mainly result from the aluminium ion (Al3 +) considering systemic effects and differences in toxicity are likely due to variation in bioavailability due to differences in the form of the ingested compound and dietary constituents.Consequently it is possible to predict the absence of effect on fertility with Aluminium ammonium (bis)sulfate by reading across from the reproductive toxicity result on Aluminium chloride basic.

Finally, under the test conditions of this study, the NOAEL for the fertility is determined at 90 mg Al3+/kg bw/day which corresponds to 1141 mg/kg bw/day of Aluminium sulphate (using the molecular mass of 342.1 g/mol for anhydrous form). Based on these results,it is possible to predict the effect following chronic exposure to Aluminium sulfate by reading across from the repeated dose toxicity result on Aluminium Chloride basic.


Short description of key information:
Under the test conditions of this study, the NOAEL for the fertility is determined at 90 mg Al3+/kg bw/day which corresponds to 1141 mg/kg bw/day of Aluminium sulphate (using the molecular mass of 342.1 g/mol for anhydrous form). Based on these results,it is possible to predict the effect following chronic exposure to Aluminium sulfate by reading across from the repeated dose toxicity result on Aluminium Chloride basic.

Justification for selection of Effect on fertility via oral route:
No guideline Study was available on Aluminum sulphate. Read- across approach using the key study performed on Aluminum chloride basic and conducted according to OECD guideline 422 in compliance with GLP is considered as adequate to assess the toxicity to reproduction of aluminium sulfate (see §"Toxicokinetics").

Effects on developmental toxicity

Description of key information
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 for aluminium sulfate as anhydrous form (molecular mass of 342.1 g/mol).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
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 study 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
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc.
- Age at study initiation: 14 - 16 weeks at breeding
- Weight at study initiation: Females: 242.5 - 333.4 g (target 160-360 grams); Males: 335.4 - 470.8 g (target 245-585 grams).
- Housing: With the exception of the breeding period and dams with litters, animals were housed individually.
- Caging:
- Before the breeding, sires were housed singly in ventilated caging.
- Dams were housed singly in conventional shoebox caging prior to breeding and during the gestation period, then with their pups during the lactation period.
- During the breeding period, sire/dam pairings were housed in wire bottomed cages.
- During lactation, pups were housed with the dam in conventional shoebox caging.
- After weaning, pups were housed individually in ventilated caging until postnatal day 120, when they were transferred to shoebox caging due to their large size.
- Harlan TEK-Fresh diamond soft bedding replaced standard corn cob bedding during the gestation and lactation periods, and also when hematuria or diarrhea was observed, or other issues as specified by the veterinarian.
- All animals received plastic enrichment tubes only for environmental enrichment.
- 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.
Details on mating procedure:
Sires and dams were allocated into breeding pairs by using SAS PROC PLAN procedure.
Animals were allowed to breed for up to five consecutive nights.
Breeding took place in wire mesh cages, which allowed vaginal plugs to collect on a tray below the cage which allowed more reliable detection of insemination.
During the breeding period, female animals were checked daily for the presence of vaginal plugs (indicating insemination). The date of breeding (i.e. insemination) was defined as the day when a vaginal plug was first detected.
Upon detection of a plug, breeding pairs were separated.
Duration of treatment / exposure:
On gestational day 6, the test item was administered to groups of pregnant animals during gestation, lactation, and to offspring during post-weaning, through to post-natal day 364 for cohort 4.

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.)
Duration of test:
1 year
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.

Beyond the treatment group allocations, dams (and their litters) were grouped according to day of delivery. This grouping allowed combining data according to postnatal day, later used in the analysis. After the end of the delivery week, litters were randomly distributed across litter groups.
Control animals:
other: A control group received water only. A citrate control group received sodium citrate at dose equimolar to citrate in the aluminium High dose group/300 mg Al/kg/Group E - 27.2 g/L.
Details on study design:
Dose 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.
Maternal examinations:
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.
Ovaries and uterine content:
No data.
Fetal examinations:
Not applicable.
Statistics:
See "any other information on materials and methods incl. tables"
Indices:
No data.
Historical control data:
No data.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
No mortality was observed in the dams during the gestation and postnatal periods in the control group, the low-dose group, the mid-dose group or the high-dose group; 20 dams were euthanized on the scheduled dates in each group. One dam that stopped nursing was euthanized early in the sodium citrate group.

Body weight
The ANOVA showed a significant effect of group (p=0.021). This was due to lower body weights in the sodium citrate group. At PND15, the mean weight of the Na-citrate group was 7.3% less than in the controls. There were no significant differences in mean body weights in dams between the aluminium-treated groups and the control group during the gestational and postnatal period.

Gestation Length
There were no statistically significant differences in gestational length between the different treatment groups.

Clinical Observations
All dams underwent daily morbidity and mortality checks during the gestational period and a clinical examination was performed on the day of delivery. Abnormal clinical observations were reported for only one dam during the gestational period.
During the postnatal period, 4 animals in the control group, 8 in the Na-citrate group, 4 in the low-dose group, 6 in the mid-dose group, and 12 in the high dose group exhibited clinical signs. Most signs were considered mild, for example alopecia and porphyrin staining. Slight dehydration was noted in 4 dams in the Na-citrate group. Diarrhoea was reported in 8 dams in the high dose aluminium group only, and thus appears to be a treatment-related effect.


Water Consumption
The table below the ranges of mean fluid consumption in mL/day (mL/kg bw/day) for the different groups for the gestation and lactation periods:

Group/Period Gestation Lactation
Control 23.0 to 31.5 (67 to 79) 35.1 to 60.6 (99 to 179)
Low Dose 35.9 to 43.7 (103 to 108) 40.1 to 60.9 (114 to 177)
Mid-Dose 42.0 to 45.2 (112 to 123) 40.9 to 69.0 (136 to 201)
High-Dose 27.4 to 31.3 (78 to 80) 39.7 to 70.2 (120 to 211)
Na-citrate 26.2 to 29.3 (66 to 76) 35.1 to 68.0 (106 to 213)

A significant effect of group was found in the ANOVA (p<0.0001). Pairwise between-group comparisons showed that the low dose group consumed significantly more water than the sodium citrate (p=0.011) and water control (p=0.0028) groups. The mid-dose group consumed significantly more than the sodium citrate (p<0.0001), water control (p<0.0001) and high dose groups (p=0.023). The differences were most marked during the gestation period.
As increased water consumption was not observed in the high dose group, the effect is not likely due to treatment.

Daily Al dosage
The target dose for the low dose group was 30 mg Al/kg bw/day, for the mid-dose 100 mg Al/kg bw/day and for the high dose 300 mg Al/kg bw/day.
Despite the deviations from the target dose, the low, medium and high dose groups showed the required trend of lowest to highest maintaining group differences in dosage.

FOB
During the gestation period, approach response, arousal, bizarre behaviour, circling, clonic convulsions, clonic convulsions rating, gait, posture, pupil response, pupil size, startle, stereotypic behaviour, tail pinch, tonic convulsions, tonic convulsions rating, total gait, tremors, tremors rating, vocalization, and writhing were zero for all dams.

The group effect (repeated measures ANOVA) for defecation (p=0.052), rearing (p=0.344), urination (p=0.487) and foot splay (p=0.089) did not reach statistical significance. A significant group effect was observed for hind limb grip strength (p=0.0047; censored analysis) driven by a lower grip strength in the Na-citrate group compared to the low and high dose groups.

During the postnatal period, bizarre behavior, circling, clonic convulsions, clonic convulsions rating, gait, posture, pupil response, stereotypic behavior, tonic convulsions, tonic convulsions rating, total gait, tremors, tremors rating, and writhing were zero for all dams.

The group effect (repeated measures ANOVA) for approach response (p=0.518), arousal (p=0.146), defecation (p=0.096), pupil size (p=0.413), rearing (p=0.151), startle (p=0.668), tail pinch (p=0.242), urination (p=0.793), vocalization (p=0.092), and foot splay (p=0.142) did not reach statistical significance. A significant across groups difference (censored analysis) was observed for forelimb grip strength (p=0.0031). Pair-wise comparisons showed that the mid-dose group was significantly less than the sodium citrate group (p=0.0005) and the high dose group (p=0.0115). The low dose group was significantly less than the sodium citrate group (p=0.012) and the control group was significantly less than the sodium citrate group (p=0.0076). The group effect for hind limb grip strength did not reach statistical significance (p=0.073) so pair-wise comparisons were not conducted.

Overall, there was no consistent effect of treatment group on any of the FOB characteristics in the dams.
Dose descriptor:
NOAEL
Effect level:
3 225 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
element
Remarks:
Al 3+
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 1 902 mg/kg bw/day
Based on:
other: Aluminium sulphate using 342.1 g/mol
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Details on embryotoxic / teratogenic effects:
Not applicable.
Abnormalities:
not specified
Developmental effects observed:
not specified

Offspring results:

Mortality

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

 

Female

Male

 

Died

Euthanized

Died

Euthanized

Control

4

4

3

1

Low-Dose

1

1

2

3

Mid-Dose

0

0

2

0

High-Dose

4

9

8

37

Na-citrate

3

2

7

3

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

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

Clinical Observations

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

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

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

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

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

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

Body Weight

Pre-weaning phase:

Analyses using the data from all cohorts combined showed no significant differences between the cohorts in body weights in the pre-weaning phase. Litter was also included in the analyses. A significant effect of litter was observed in both male and female pups.

Results of pair-wise comparisons between treatment groups in the female pups, showed that Na-citrate and high dose groups had significantly lower pre-weaning body weights than the control and low-dose groups (low dose v Na-citrate, p=0.0007; low dose v high dose, p=0.0398; control v Na-citrate, p<0.0001; control v high dose, p=0.0072).

In the male pups, the low dose group had significantly greater body weights than the Na-citrate group (p=0.0004) and the high dose group (p=0.0239). The control group mean body weights were significantly greater than the Na-citrate group (p<0.0001) and also significantly greater than the high dose group (p=0.0051). The mid-dose group mean body weight was significantly greater than the Na-citrate group (p=0.0405).

Post-weaning phase:

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

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

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

Day 64 cohort, females: High dose females were significantly lighter than all the other dose groups. The group x Study Day interaction term was significant. On Study Days 43 and 56, the high dose group was significantly lighter than all the other groups.

Day 64 cohort, males: High dose males were significantly lighter than all the other dose groups. The Na-citrate group was significantly lighter than the low dose and the control groups (p=0.0008, p<0.0001, respectively). The group x Study Day interaction term was significant. On Study Day 43, the high dose group was significantly lighter than all the other treatment groups (all p<0.0001). The Na-citrate group was also lighter than the control group (p=0.0184) on this day. On Study Day 56, the high dose group was significantly lighter than all the other treatment groups (all p<0.0001); the mid-dose group was also significantly lighter than the control group (p=0.0211). The Na-citrate group was significantly lighter than the low dose (p<0.0001) and mid-dose (p=0.0003) groups on this study day also.

Day 120 cohort, females: The effect of group was significant (p<0.0001) and pair-wise comparisons showed that the high dose group was significantly lighter than all the other groups (p <0.0001, p=0.0002, p=0.0151, and p=0.0002 for comparisons with the control, low-dose, mid-dose and Na-citrate groups, respectively).

Day 120 cohort, males: The effect of group was significant (p<0.0001) and pair-wise comparisons showed that the Na-citrate group and mid-dose groups were significantly lighter than the control group (p=0.0011 and p=0.0016, respectively). The Na-citrate group was also significantly lighter than the low dose group (p=0.0203). Pre-dose body weight was included as a covariate in the analyses. The Group x Study Day interaction term was significant. In pair-wise comparisons, the high dose group was significantly lighter than the other treatment groups on Study Day 43, 56, 70, and 84. The Na-citrate and mid-dose groups were significantly lighter than the control group on Study Days 70, 84 and 98.

Day 364 cohort, females: The effect of group was significant (p=0.0008) and pair-wise comparisons showed that the high dose group was significantly lighter than the control and mid-dose groups (p=0.0015 and p=0.0032, respectively) but not the low dose group. The group x Study Day interaction term was significant. The high dose group was significantly lighter than the control group on Study Days 294, 308, 322, 336, 350 and 364. The Na-citrate group was significantly lighter than the control on Study Days 322, 336, 350 and 364.

Day 364 cohort, males [note: males euthanized at Day 84]: The effect of group was significant (p=0.001) but there were no significant pair-wise differences between the control, low-dose, mid-dose, and Na-citrate groups. The group x Study Day interaction term was significant. Pair-wise comparisons showed that the high dose group was significantly lighter than the control and low-dose groups (p=0.0027 and p=0.0016, respectively) on Study Day 70. On Study Day 84, the high dose group was significantly lighter than the control, low-dose and Na-citrate groups.

The results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. An effect of Na-citrate was observed in the female pups.

Growth Curve Parameters

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

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

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

Water Consumption

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

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

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

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

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

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

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

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

Fluid consumption varied significantly between study days. In mid-dose males (Day 364 cohort), the mean fluid consumption during the first post-weaning week was 16.0 mL/day (equivalent to 171 mL/kg bw/day; 33% greater than in the controls); on study day 70 it was 36.4 mL/day (equivalent to 93 mL/kg bw/day; 63% greater than in the controls) and decreased on a per body weight basis until the end of the study. In high-dose females (Day 364 cohort), the mean fluid consumption during the first post-weaning week was 16.3 mL/day (equivalent to 207 mL/kg bw/day; 60% greater than the controls); on study day 112 it was 37.6 mL/day (equivalent to 130 mL/kg bw/day; 124% greater than the controls) and decreased on a per body weight basis until the end of the study.

Overall, dosing of animals with aluminium citrate led to an increase in fluid consumption compared with the control animals.

Dosing with Na-citrate was associated with a significant increase in fluid consumption relative to the controls in most cohorts, with the exception of the Day 64 cohort females (fluid consumption was significantly lower in the Na-citrate group) and the Day 364 males (no significant difference between the two groups).

The animals’ fluid consumption varied with time and, in mature animals, was less than expected (120 mL/kg bw/day) with implications for the actual dosage of test item received.

Actual Doses Received

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

Males

Group

Week 1

Week 7

Week 14

Week 28

Week 49

Low-Dose

134%

57%

37%

20%

17%

Mid-Dose

174%

84%

51%

28%

23%

High-Dose

165%

117%

-

-

-

Females

Low-Dose

145%

60%

57%

34%

33%

Mid-Dose

199%

74%

64%

38%

41%

High-Dose

205%

118%

93%

58%

42%

Despite the deviations from the target dose, the low-, mid- and high-dose groups showed the required trend of lowest to highest maintaining statistically significant group differences in dosage. For the majority of the study period, the actual dose received was less than the target dose in all treatment groups.

Organ Weight

Brain weights.

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

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

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

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

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

Pathomorphology and Histology

Necropsy Results

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

The numbers of rats per cohort and treatment group that exhibited urinary tract pathology (hydronephrosis, ureteral dilation, obstruction and/or presence of calculi) are provided in the tables below (data extracted from Table 4 of the final report):

Females

Day 23

Day 64

Day 120

Day 364

Control

0

1

0

0

Low-Dose

0

0

0

0

Mid-Dose

0

1

0

0

High-Dose

0

3

2

3

Na-citrate

0

0

1

0

Males

Day 23

Day 64

Day 120

Day 364

Control

0

0

0

0

Low-Dose

0

0

0

1

Mid-Dose

0

3

1

0

High-Dose

0

11

7

5

Na-citrate

0

1

0

0

Urinary tract pathology was a treatment-related effect.

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

Histopathological examination of CNS tissue and muscle (microscopic)

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

Day 64 cohort:

Control group – one male rat showed very mild inflammation of connective tissue around the sciatic nerve.

Low dose group - All tissues were normal.

Mid-dose group - All tissues were normal.

High dose group - All tissues were normal.

Na-citrate group - All tissues were normal.

Day 120 cohort:

Control group – All tissues normal.

Low-dose group - All tissues were normal.

Mid-dose group - All tissues were normal.

High-dose group - All tissues were normal.

Na-citrate group - All tissues were normal.

Day 364 cohort:

Control group – 3 females and 2 males had low numbers of neurons in the thoracic dorsal root ganglion, the neurons had small vacuoles.

Low dose group - 1 female had a focal area of gliosis at one edge of the hippocampus; 4 female and 2 male rats had small numbers of neurons in the sections of thoracic dorsal root ganglion with small vacuoles in the cytoplasm.

Mid-dose group – 3 females and 1 male had low numbers of neurons in thoracic dorsal root ganglion section and the neurons had vacuoles; a male had astrocytoma in the posterior hippocampus and 1 male had gliosis in one side of the central canal.

High dose group - 3 female rats had low numbers of vacuolated neurons in the thoracic dorsal root ganglion; a vacuolated neuron was also observed in a lumbar spinal cord section from one rat, and from a section of cervical ganglion in another rat.

Na-citrate group – 3 females and 2 males had low numbers of neurons in the thoracic dorsal root ganglion section and the neurons had vacuoles; 1 male rat had occasional spheroids in the white matter of the lumbar spinal cord.

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

Group

Sex

Day 364

Control

M

2

 

F

3

Low-Dose

M

2

 

F

4

Mid-Dose

M

1

 

F

3

High-Dose

M

n/a

 

F

3

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

Developmental Landmarks

Females

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

Males

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

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

FOB (neonatal pups)

Females

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

Males

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

FOB (juveniles)

Day 364 cohort

Females

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

Males

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

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

FOB (adult pups)

Day 364 cohort

Females

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

FOB Parameter Group effect Pairwise Differences

Wasting P=0.0040 High dose group had sig. more wasting than low dose group (p=0.0308), mid-dose group (p=0.0213) and controls (p=0.0042)

Na-citrate group had sig. more wasting than low dose group (p=0.0345), mid-dose group (p=0.0233) and controls (p=0.0044).

- treatment-related effect

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

Mouth and nose deposits P<0.0002 High dose group had sig. more than controls and mid-dose group, but low dose and controls had sig. more than mid-dose group also. Not consistent with a treatment-related effect.

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

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

Arousal (open field) P=0.0011 The high dose group exhibited more arousal than the low dose group, the controls, and the Na-citrate group. The low-dose and mid-dose groups showed sig. more arousal than the controls.

Defecation P<0.0001 The high and mid-dose groups have more faecal boluses than the low-dose group, the controls and also the Na-citrate group. Likely a treatment-related effect.

Defecation characteristics P<0.0001 As above

Pupil response P<0.0001 The high dose group lacked response compared to the control and mid-dose groups. The low-dose and mid-dose groups lacked response compared with the control. The Na-citrate group also lacked response compared to the control.

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

Rearing P<0.0001 All of the treatment groups exhibited significantly more rears compared with the controls. The low-dose group exhibited sig. more rears than the high dose group. Not consistent with a treatment-related effect.

Tail pinch P=0.0001 The mid-dose group had sig. more abnormal reaction than the low dose, mid-dose, high dose and Na-citrate groups. The low dose group had sig. more abnormal reaction than the control group. Overall, not clearly consistent with a treatment-related effect.

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

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

Foot-splay P<0.0001 The low-dose group had sig. greater foot-splay measurements than the high dose group, the mid-dose group and the Na-citrate group. The control group had significantly greater foot-splay than the mid-dose group, the high-dose group and the Na-citrate group. Weak evidence of dose-response and a treatment-related effect.

Forelimb grip strength P<0.0001 The controls had sig. greater forelimb grip strength than the mid- dose group (p<0.0001), the high-dose group (p=0.0066) and the Na-citrate group (p=0.0101). The low-dose group had sig. greater forelimb grip strength than the mid-dose group (p=0.0085). Some evidence of dose-response; treatment-related effect.

Hind-limb grip strength P<0.0001 The controls had sig. greater forelimb grip strength than the mid- dose group (p=0.0007), the high-dose group (p<0.0001) and the Na-citrate group (p<0.0001). The low-dose group had sig. greater forelimb grip strength than the mid- dose group (p=0.0093), the high-dose group (p<0.0001) and the Na-citrate group (p=0.0012). Some evidence of dose response; treatment related effect.

Males

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

FOB Parameter Group effect Pairwise Differences

Fur appearance P<0.0001 High-dose group had sig. more abnormal appearance than controls (p=0.0169), low-dose group (p=0.0016), and mid-dose group (p=0.0185).

Mouth and nose deposits P=0.0216 High-dose group had sig. more deposits than the low-dose group and the mid-dose group.

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

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

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

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

Defecation P<0.0001 The Al-treated groups and the controls had more faecal boluses than the Na-citrate group. The low-dose group had fewer boluses than the controls, mid-dose group, and the high dose group. Not consistent with a treatment-related effect.

Defecation characteristics P<0.0001 Not clearly related to treatment.

Rearing P<0.0001 The high dose group exhibited sig. fewer rears than the Na-citrate group. The mid-dose group exhibited sig. more rears than the control and the low-dose groups. The low-dose group exhibited sig. more rears than the control group. Variable and not clearly consistent with a treatment-related effect.

Tail pinch P=0.003 The control group and the mid-dose groups had significantly more abnormal responses than the high dose group. The Na-citrate group had significantly more abnormal responses than the controls, the low-dose and the mid-dose groups. Not consistent with a treatment-related effect.

Urination P<0.0001 The high dose group had fewer urine pools than the mid-dose group, The Na-citrate group had more urine pools than the low-dose group and fewer urine pools than the mid-dose group. Overall, not consistent with a treatment-related effect.

Urine characteristics P<0.0001 Not clearly related to treatment.

Foot-splay P=0.0004 The low-dose group showed sig. greater foot-splay than the mid-dose group and the Na-citrate group.

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

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

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

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

Motor Activity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Auditory Startle Response

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

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

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

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

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

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

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

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

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

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

T-maze

The T-maze testing was conducted at PND 21.

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

Group

Male

Female

Control

42.11

26.32

Low-Dose

25.00

42.11

Mid-Dose

31.58

47.37

High-Dose

63.16

31.25

Na-citrate

26.32

50.00

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

Morris Water Maze

Training Trial Latencies

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

Platform-Removed Probe Test Search Strategies

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

Platform Visible Latencies

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

Platform Visible Type of Search

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

 

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

Haematology

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

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

Day 23 cohort:

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

Day 64 cohort:

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

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

the low-dose and the mid-dose groups.

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

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

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

The anaemia had resolved in the females.

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

Coagulation parameters:

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

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 damsad libitumviadrinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.

 

Endpoints and observations in the dams included water consumption, body weight, a Functional Observational Battery (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 assessedca.PND 22. In the adult pups, the data provide little evidence for an Al effect on the autonomic function domain, the sensimotor function domain, or excitability. Significant wasting (physiological domain), was observed in the high dose females and appears related to treatment. Characteristics of defecation (number of boluses) also showed differences with treatment. In addition, there was limited evidence of effects on activity/well-being of the pups at the high dose as reflected in fur appearance, deposits and rearing. There was some evidence for dose-response relationships between neuromuscular measurements – hind-limb and fore-limb grip strength and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. Although the 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 many strengths. It was conducted according to GLP with a design based on OECD TG #426. The study used adequate numbers of animals and randomization to reduce bias, assessed endpoints in both female and male offspring, and studied a wide range of neurotoxicity endpoints. Haematology, clinical chemistry, pathology and general toxicity endpoints were also assessed. Three dose levels were used although the highest was close to the 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).Asthe offspring were dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. Urinary tract pathology was observed in rats in the high dose group, more frequently and more severe in the males. The study showed no evidence of an effect of Al-citrate on memory or learning but a more consistent effect was observed in endpoints in the neuromuscular domain.

 

The ambiguity as to the critical period of exposure and the time-varying water consumption complicate the derivation of a point-of-departure from this study. A LOAEL of 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).
Effect on developmental toxicity: via oral route
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 developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In terms of hazard assessment of toxic effects, available data on the toxicity to reproduction/development of other aluminium compounds was taken into account by read-across following a structural analogue approach, since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al3 +) as described in the toxicokinetics section.

No developmental toxicity studies conducted on aluminium sulfate in mice and rats have been identifed in the literature. However, several developmental toxicity studies in animals have shown that oral gestational exposure to Aluminium via aluminium salts (e.g. aluminium nitrate, aluminium hydroxyde, aluminium lactate, etc) may induce developmental effects, among them neurodevelopmental effects in rats and mice. It must be highlighted that these effects have been observed under conditions that enhanced aluminium uptake, particularly maternal intake of compounds that are highly bioavailable (e.g. Aluminium citrate and nitrate), concurrent exposure to dietary constituents that contribute to increased absorption of Aluminium (e.g. citrate), and/or bolus administration by gavage which could result in higher blood levels than dietary administration or administration via the drinking water. Moreover very high dosages were used (ca. 200 to 2000x normal human exposure). Part of the reason for using such high dosages was the low solubility and bioavailability of some aluminium compounds and the limited sensitivity of available analytical methods to determine small changes from endogenous levels of aluminium. Moreover, the achieved dose of aluminium in maternal plasma was not measured in the majority of case. In the studies with aluminium administered in the diet or drinking water, dosages were generally identified in terms of the target dose, e. g. 1000 µg Al/g diet, without calculation of the actual dose administered based on the food or water consumption. Further, for the majority of the studies, there was no assessment of the background levels of aluminium in the food and water provided for the animals.

These factors generally lead to the conclusion that the dosages used in developmental toxicity studies up to date have been much greater than those that would be encountered in the human consumer or worker situation. In addition, the actual dose administered has usually been under-estimated because background aluminium levels in the diet and drinking water provided for the animals have not been taken into account.

Consequently, a recent combined one-year developmental and chronic neurotoxicity study with Al-citrate was conducted to determine whether aluminium administered at near toxic concetnrations 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 (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.

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 for aluminium sulfate as anhydrous form (molecular mass of 342.1 g/mol).


Justification for selection of Effect on developmental toxicity: via oral route:
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 developmental toxicity of aluminium sulfate (see §"Toxicokinetics").

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available for the toxicity to reproduction by oral and dermal route , by inhalation for Aluminium sulphate.

Self classification:

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

Aluminium sulphate is not classified for the lactation according to the Regulation (EC) No. 1272/2008 and the Directive 67/548/EEC criteria.