Bis(acetato-O)hydroxyaluminium

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A NOAEL of 30 mg Al/kg bw/d has been determined in a chronic developmental neurotoxicity study for the source substance (reference [7.5.1 -1]). Considering the differing molecular weights of source and target substance, a NOAEL of 180 mg/kg bw/d was determined for aluminium acetate (reference [7.5.1 -2]).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental neurotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 426 (Developmental Neurotoxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Animals were housed singly in shoebox or ventilated cages except during breeding (wire-bottomed cages) and until weaning (grouped with rest of litter) for the pups.
- Diet: Rats were fed AIN-93G growth food (Purina TestDiet) until PND 95–99, followed by AIN-93M maintenance food (Purina TestDiet) for the duration of the study; ad libitum).
- Water: ad libitum
- Acclimation period: The acclimation period lasted 9 days.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose materials (aluminum citrate) were calculated and prepared from three different lots from Chemos GmbH, Regenstauf, Germany. The aluminum content by mass was 9.3%, 9.8% and 8.7% respectively based on the manufacturer product sheet. Fresh filtered solutions were prepared weekly using de-ionized
water and the pH was adjusted between 6 and 7.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Details on mating procedure:

- Length of cohabitation: Five consecutive nights
- Proof of pregnancy: vaginal plug

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The final concentrations of Al for each of the doses have been independently assessed using inductively coupled plasma mass spectrometric analyses of metal concentrations by Maxxam Analytics Inc. (Burnaby, Canada) using certified GLP.

Duration of treatment / exposure:

From gestational day 6 the test item was administered to groups of pregnant animals during gestation and throughout lactation to postnatal day 21 (PND). The offspring was further exposed during post-weaning, through to postnatal day PND 364.

Frequency of treatment:

Daily

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Dams: 20/group
Pups: 80 females/group, 80 males/group

Control animals:

yes, concurrent no treatment

Details on study design:

Animals were segregated into breeding pairs and allowed the opportunity to breed for up to five consecutive nights. 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.
- Dose selection rationale: Doses were selected based on the results of a previous 90-day pilot toxicity study with aluminium citrate and, the maximum solubility of aluminium citrate in water (high dose).

Positive control:

A group of animals exposed to sodium citrate at a citrate concentration equimolar to the highest aluminium citrate solution (27.2 g/L) was included to investigate the possibility of counter ion effects.

Parental animals: Observations and examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Gestational day 6, 13, 20; Postnatal day 8, 15 and 22

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included morbidity and mortality.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on gestational days 7 and 13 and on PNDs 3 and 10
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: vaginal opening for females starting on PND 26 and preputial separation for males starting on PND 35.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included morbidity and mortality.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Not specified
- How many animals: all pups
- Parameters checked: mean cell volume (MCV), red blood cells (RBC), platelet count, hematocrit (HCT), mean cell hemoglobin (MCH), hemoglobin (HGB), white blood cells (WBC), absolute granulocytes (ABS_GRAN), absolute agranulocytes (ABS_AGRAN)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Animals fasted: Not specified
- How many animals: all pups
- Parameters checked: albumin/ globulin ratio (A_G), albumin (ALB), Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), calcium (CA), chloride (CL), creatinine (CRE), Globulins (GLOB), glutathione (GLU), potassium (K), sodium (NA), PHOS, Total biliubin (TBIL), Triglycerides (TG), Total protein (TP), urea (UREA)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
FOB: One male and one female pup from each litter (pups 4 and 8, which were allocated to the day 364 group) underwent FOB examinations on PNDs 5, 11, 22, 36, 45, 56, and biweekly thereafter until the week of PND 350. T-maze tests were conducted on PND 22. The Morris water maze tests were conducted prior to PNDs 59, 61, 63 and 64, PNDs 114, 116, 118 and 119, and PNDs 357, 359, 361 and 362. Motor activity was monitored on PNDs 15 or 16, 17, 21, 62, 117 and 363 as per OECD guidelines 426.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: All surviving animals [after weaning of the offspring]

Postmortem examinations (offspring):

SACRIFICE
- Offspring: all surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The brain was prepared for microscopic examination and weighed, respectively.

Statistics:

Statistical analyses were conducted using SAS® Release 9.1 for Windows. Data collected on dams and pups was analyzed separately and all statistical analysis on the pups was performed separately for each sex. Analyses for pups were done separately for each test day group unless specified otherwise. Statistical significance was declared when P < 0.05. All statistical analyses were done based on the principle of intention-to-treat.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs were observed in 4, 4, 6 and 12 animals of control, 30, 100 and 300 mg/kg bw/d group, respectively. Most of the findings included mild dermatological leasions (alopecia and porphyrin staining) or mild stress related symptoms. Eight animals of the high dose group showed diarrhea.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences between mean body weights of controls and treated groups.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

During gestation and lactation, low- and mid-dose animals consumed significantly more fluid than controls during weeks 1 and 2 (gestation days 6–20). For the control group, mean fluid consumption during gestation varied between 23.0 and 31.5 mL per day, or 67–79 mL per kg body weight per day (note: 120 mL per kg per day was the benchmark used for designing the study, mL/kg body weight was determined from the weekly mean fluid consumption/the weekly mean body weight in kg). During lactation, mean fluid consumption was between 35.1 and 60.6 mL per day, or 99–179 mL per kg per day. For the mid-dose group, mean fluid consumption during gestation varied between 42.0 and 45.2 mL per day, or 112–123 mL per kg body weight per day. During lactation, mean fluid consumption was between 40.9 and 69.0 mL per day, or 136–201 mL per kg per day. For the high-dose group (not significantly different from controls), mean fluid consumption during gestation varied between 27.4 and 31.3 mL per day, or 78–80 mL per kg body weight per day. During lactation, mean fluid consumption was between 39.7 and 70.2 mL per day, or 120–211 mL per kg per day. Overall, concentrations of Al citrate at the low- and middose concentrations were associated with a significant increase in fluid consumption in pregnant and lactating rats; however, interpretation is complicated by a lack of consistent effect in the high-dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

element

Remarks:

aluminium

Sex:

female

Basis for effect level:

water consumption and compound intake

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In the day 23 group, a few animals in the high-dose group were observed to have abdominal distention or were small and cold. In the day 64 group, three Na citrate group animals were observed to be thin with poor hair coats. One was euthanized. One control group female was bloated, had diarrhea and was later euthanized. In the high-dose group, one female and seven males were thin, had diarrhea, mild dehydration and poor hair coat.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A number of animals from each treatment group and sacrifice day group died or were euthanized prior to their planned sacrifice dates. A number of animals from each treatment group and sacrifice day group died or were euthanized prior to their planned sacrifice dates. The main finding due to the experimental treatment mentioned by the pathologist was the presence of urinary tract lesions of hydronephrosis, ureteral dilation, obstruction and/or presence of calculi. The calculi were chalky white concretions and deposits varying from sand-like material through to large, irregular stones measuring up to 4 mm diameter, found in the urinary system of many test animals. Obstruction of the urinary tract by calculi occurred in many affected animals, especially the high-dose males. Obstruction was found to occur at any level of the urinary tract, including the urethra, bladder, ureters, and renal pelvis. Dilation of the ureters and hydronephrosis were common pathologic findings in both partially and completely obstructed rats. Several rats died, presumably due to hyperkalemia. This was especially prevalent in the high-dose group males, where urinary calculi blocked the urinary tract at various levels, but also a few animals from the mid-dose group and a few random ones from other groups also had urinary tract lesions.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the pre-weaning phase (birth to PND 22), mean body weights of male and female Na citrate and high-dose groups were significantly lower than controls and low-dose groups. The Na citrate group was also significantly lower than the low and mid-dose groups. At post-weaning no significant differences in male mean body weights of Na citrate, low-dose or mid dose groups compared with controls in day 364 group, which covered the entire study period. High-dose males were terminated from study day 98 onwards because of excessive clinical signs (including weight loss), and were not included in the repeated measures ANOVA after study day 98. The only significant effects on body weights occurred in the Na citrate and high-dose treatment groups: At the time of weaning, male pups of the Na citrate group had 17% lower body weights relative to controls, compared with 12% lower weights in high-dose males. Following weaning, the Na citrate males recovered but the high-dose males continued to lose weight. By PND 84 the mean weight of male high-dose pups was 30% below controls, at which time they were euthanized. By comparison, the Na citrate mean weight was 9% (not significant) below controls on day 84. The high dose of Al citrate is therefore considered to have had an adverse effect on body weights of male pups.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

For the day 64 group males, the fluid consumption of mid-dose animals was significantly greater than the rest. As well, consumption by the high dose males was greater than either the low-dose or control groups and that of the Na citrate group exceeded control group consumption. For the day 120 group males, the fluid consumption of the mid-dose group exceeded that of the controls, low and Na citrate groups and the Na citrate group fluid consumption exceeded that of the controls. For the day 364 group males, during the study period, the fluid consumption of mid dose and Na citrate group males was significantly greater than that of the controls and the low dose group. High-dose males were euthanized on study day 98 because of excessive clinical signs (including weight loss), therefore data were unavailable thereafter, and high-dose group male fluid consumption data were not included in the repeated measures ANOVA. For the day 364 group mid-dose group (other groups followed a similar pattern), mean fluid consumption during the first post-weaning week was 16.0 ml per day (171 mL per kg body weight per day) compared with 13.7 mL per day (129 mL per kg per day) for controls, a difference of 17% (33% on a per body weight basis). The largest difference was at day 70 when mid-dose males had fluid consumption that was 36.4 ml per day (93 ml per kg per day) compared with 26.0 (57 mL per kg per day) for controls, a difference of 40% (63% on a per body weight basis). Differences between mid-dose and control groups were less thereafter. From about day 70 to the end of the study, mean fluid consumption of all groups declined gradually. For example, from PND 126 through to PND 364, mean fluid consumption of the mid-dose group fell from about 30 to about 20 mL per day. Since body weight rose significantly during this period, the result was a decrease in fluid consumption on a per body weight basis. At PNDs 112, 238 and 364, mean fluid consumption of mid-dose males was 65, 30 and 26 mL per kg per day, respectively. This compared with controls having consumptions of 48, 22 and 20 mL per kg per day. For the day 64 group females, fluid consumption of the high-dose group was greater than the rest, that of the mid-dose group was greater than fluid consumption of the low-dose and control groups, and also that of the Na citrate group was lower than consumption by the controls. For the day 364 group females, during the study period, the mean fluid consumption of the high-dose group exceeded that of the rest, while that of the controls was lower than the rest. The low-dose group had lower fluid consumption than the Na citrate, mid- and high-dose groups. At specific time points: the fluid consumption of high-dose females was significantly greater than controls on PNDs 43 through 210.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In day 23 group females, mean cell volume (MCV) in the control group was significantly greater than in the low-dose group. As well, nucleated red blood cells were different in the low than in the control, mid or high-dose groups and platelet counts were less in the low-dose group than in the high-dose group. In day 23 group males, MCV in the high-dose group was significantly less than in the control group. For most parameters, except mean cell volume, there were no statistically significant differences across groups so these observations are not related to treatment effects. In the day 23 group females and males, the mean cell volume differences are not deemed to be clinically significant. In day 64 group females, the high-dose group hematocrit (HCT), mean cell hemoglobin (MCH) and MCV were all lower than in the other four groups. Hemoglobin (HGB) was lower in the high-dose group than in the mid-dose, low-dose or control groups. White blood cells (WBC) were higher in the high-dose group than in the control or low-dose groups; however, absolute agranulocyte counts were less in the high-dose group than in the controls. In day 64 group males, MCH and MCV were lower in the high-dose group than in the rest. HCT was less in the high-dose group than in the control and low-dose groups. Red blood cell counts were slightly higher in the high-dose group than in the mid-dose or sodium citrate groups. In day 120 group females, MCV in the high-dose group is less than in the other four groups. MCH in the high-dose group is slightly less than in the mid-dose, Na citrate and control groups. WBC, absolute granulocytes (ABS_GRAN) and absolute agranulocytes (ABS_AGRAN) in the high-dose group are higher than in the other Al citrate groups and controls. In day 120 group males, no Al citrate treatment-related differences were seen, however by this milestone, all of the high-dose group males had been euthanized due to renal calculi interfering with kidney function. In the day 364 group females and males, no statistically significant differences were seen in the hematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phosphatase was slightly elevated in the high-dose group in the day 23 group males, day 64 group males and females and day 120 group females. Serum calcium was elevated in the high-dose group in the day 23 group, 2 and 3 females and day 64 group males. Serum phosphate was lower in the high-dose day 23 group females than in the controls and higher in the high-dose day 64 group males than in the rest of the groups. As expected, sodium was slightly elevated in the Na citrate groups in day 23 group males and females and slightly lower in the high-dose group of day 64 group males than in the corresponding mid, low and control groups. It was slightly higher in the mid-dose group than in the Na citrate and high-dose groups of day 64 group females. Chloride was lower in the high-dose group of day 64 group males than in the rest, and was slightly lower in the Na citrate groups of day 120 and 364 group females than in the controls and also the low-dose group of day 364 group. A number of the differences observed in the high-dose group may be attributed either primarily or secondarily to the effects of aluminum exposure. Serum chemistry features of aluminum toxicity include elevated alkaline phosphatase and serum calcium levels, and both of these were observed, especially in the day 64 group animals. The high-dose animals of both sexes were less robust and it is no surprise that parameters such as total protein, albumin and globulin were slightly lower in especially day 64 group, and this is also a feature of high aluminum exposure. Creatinine and urea elevations in the day 64 group males may be due to renal calculi, a high incidence of which was encountered in high-dose male animals, leading to the deaths of several animals and the sacrifice of all of the remaining high-dose males before the day 120 milestone. Differences in electrolyte levels (serum sodium and chloride) in day 23 group animals are likely due to the high sodium load in the Na citrate reference group. Differences in the later sacrifice day groups were not pronounced and there are no obvious explanations other than aluminum toxicity in the high-dose Al citrate groups and high sodium load in the Na citrate group.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

High-dose group females and males had significantly greater days to reach landmarks compared with the other groups. The Na citrate females and males had significantly greater days to reach landmarks as compared with the other groups except for the high-dose group. The differences in days to reach landmarks in some litters suggests a degree of heritability of this trait, however all of these litters were from the high-dose treatment group so this is almost certainly a treatment effect.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Similarly to the macroscopic findings in the animals undergoing unscheduled necropsies, the main gross postmortem finding was the presence of white precipitates in the urinary tracts of many animals, both male and female. Secondary pathologies such as hydronephrosis and ureteral dilation occurred in most affected animals. This was most common in high dose group animals, especially males. A significant number of males with renal calculi died or were euthanized before their scheduled necropsy date, and for animal welfare reasons, all remaining high-dose group males from Day 120 and 364 groups were euthanized on between PNDs 82 and 86. Only six high-dose group males from each of Day 120 and 364 groups had no gross postmortem findings. A second finding in some high-dose group animals, especially males (five from day 64 group, two from day 120 group and two from day 364 group), but also one female from each of Day 23 and 364 groups and one day 364 group male from the Na citrate group, was fluid colonic content. The main gross postmortem finding of urinary tract pathology, principally in the high-dose group, was interpreted as a treatment related finding, as was the fluid colonic contents in some animals. The test item clearly precipitated in the urinary tract causing stone formation and blockage, and resulted in fluid colonic content, but did not have any anatomic-pathologic effects on other organs.

Histopathological findings:

no effects observed

Description (incidence and severity):

In Day 23, 64 and 120 groups, there were no abnormal microscopic findings except for one female in day 23 group, low-dose group, that had both a necrotic neuron and a neuron with satellitosis in the section of basal ganglia, and a day 64 group, control group female with very mild inflammation of the connective tissue around the sciatic nerve. In day 364 group, a proportion of the animals had neuronal cytoplasmic vacuolation in mainly the thoracic, but also some lumbar dorsal root ganglia. Cortical and hippocampal areas were found to be free of pathology.

Other effects:

no effects observed

Description (incidence and severity):

Motor activity:
Mean ambulatory counts declined in general from the 1st to the 12th interval, and from day 64 group to day 364 group, the decline became slightly more pronounced in both sexes. However, there were no consistent patterns of significant group differences over the course of the study and when differences occurred, they were mild. The high-dose group of day 64 group males had low ambulatory counts (indicating less active animals) in later intervals, and this may be a mild treatment-related effect; however, this observation was not repeated in other sacrifice day groups. Both treatment effects appear to have resolved over time as they were not repeated in later sacrifice day groups.

T-maze
Spontaneous alternation was defined as initial entry into the previously blocked arm of the maze, during the second trial. The rates of alternation were not statistically significantly different among the groups. If spontaneous alternation were to occur purely by chance, alternation rates would be expected to be 50%, however, in previous studies alternation rates slightly higher than 50% were observed in rat pups of weanling age but alternation rates of lower than 50% were observed in younger animals that seemed to explore more cautiously. The only group in this study that alternated at a rate greater than 50% was the high-dose males. The reason for this is not known. The overall rate of alternation in this study is less than 50%.

Morris water maze
In the training trials, latencies tended to decrease trial over trial within a test day (i.e. Trials 1–4, 5–8, 9–12) and between test days (Trials 1, 5, 9) in all groups, sexes and sacrifice day groups. There were no significant differences among groups in either sex in any of the sacrifice day groups. In the platform removed probe tests, rats tended to use focal correct search strategies in a minority of instances, and in general, were more inclined to use focal incorrect, scanning and random strategies more frequently. Thigmotaxis was rarely observed. There were no significant group-wise differences in the platform-removed probe tests. In the visible platform probe tests, mean latencies ranged from 10.8 to 27.9 s for the first trial and from 6.2 to 16.5 s for Trial 2. There were no group-wise differences in the latencies or the search strategies in the visible platform test. Overall the indirect search strategy was used more than half the time, while the direct search strategy was used somewhat less than half the time. There were no significant group-wise differences on the visible platform probe tests.

Comparative bioavailability for five aluminium salts
In drinking water, the concentrations of all analytes (aluminum, manganese, iron, zinc, copper) were near to or below the limit of quantification. For aluminum, the concentration was 2 ng/mL. All analytes in the control dosing solutions were below the limits of quantification. The aluminum concentration in the diet was measured at 9 µg/g. The nominal concentration of aluminum in the dosing solutions for the different salts was 3 mg/mL. All animals survived without any visible signs of clinical deterioration or toxicity up to scheduled euthanasia at day 7 or 14. There were no significant differences in body weights between groups for males and females in the 7 day exposure group (males P=0.9202; females P=0.4216). There were no significant differences between groups for the 14-day exposure females (P=0.6774) and males (P=0.1652). There were no significant group differences in food consumption of 14 day exposure males (P=0.1871). However, for the 14 day exposure females, there was a significant difference between groups (P=0.0543). Specifically, the females receiving aluminum nitrate had a significantly higher (+18%) daily food consumption compared with the control group (P=0.0262). While average daily food consumption in the males significantly increased over the time period between 7 and 14 days of exposure to treatments (P<0.0001), the females did not consume significantly more over the same interval (P=0.5920). There were no significant differences in water consumption between groups in either the males (P=0.6414) or females (P=0.8577) for the 14 day exposures. There were no gross postmortem abnormalities in any of the animals in the study. In essence, very few statistically significant differences in aluminum concentration existed between treatments for any tissue of either sex. The tissues where differences did exist included liver (day 14 males), kidney (day seven males and day 14 females), and bone (day seven males), although for liver the difference is between treatments, not between controls and treated groups. Interestingly, comparisons of aluminum concentrations at day 14 and 7 reveal that, for all treatments, most tissues exhibited a decrease in median aluminum concentration between day 7 and 14.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant group differences were recorded for the neonatal rat pups of either sex. However, significant differences emerge over time in a subset of adult animals.
Females: No abnormal observations were detected for tonic convulsions (home cage), clonic convulsions (home cage), tremors (home cage and open field), posture (home cage and open field), conjunctivitis (handling observations) or total gait (open field). Some individual variability (but no significant group difference) were noticed for palpebral closure, lacrimation, red crusty deposits (eye), ocular exudate, 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. Non-normal observations with significant group differences include wasting, fur appearance, mouth and nose deposits, eye opacity, salivation, arousal, defecation, defecation characteristics, pupil response, rearing, pupil size, tail pinch, urination, and urine characteristics.
Males: No non-normal observations were found 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), palpebral closure (home cage), conjunctivitis (handling observations), ocular exudate (handling observations), writhing (handling observations). Some non-normal observations but no significant group differences were found in 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. Non-normal observations with significant group differences include fur appearance, mouth and nose deposits, eye opacity, red crusty deposits, exophthalmus, piloerection, defecation, defecation characteristics, tail pinch, rearing, urination, and urine characteristics.
In summary, high dosage Al exposure did not seem to be associated with autonomic or sensimotor dysfunction. There was, however, a weak association between high dosage Al exposure and reduced home cage activity, excitability.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day

Based on:

element

Remarks:

aluminium

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Results for P0 and F1 descriptions were included as cited in Poirier et al. (2011).

Conclusions:

Based on the results of the above study a LOAEL of 100 mg Al/kg bw/d and a NOAEL of 30 mg Al/kg bw/d was determined for Sprague Dawley rats.

Executive summary:

In a chronic developmental neurotoxicity study in accordance to OECD 426, aluminium citrate was administered to pregnant Sprague Dawley rats via drinking water. Treatment started on gestational day (GD) 6. The concentrations administered were aimed to deliver aluminium doses of 30, 100 and 300 mg/kg bw/d based on an expected daily water intake of 120 mL/kg bw/d. The treatment doses varied in the different phases due to changes in the water intake over time. During gestation the target dose was almost achieved at the low dose. During lactation the treatment dose for the dams was higher than 30 mg/kg bw/d (around 40 mg/kg bw/d). The mean dose was 40.2 and 43.5 mg (again higher than 30) for male and female pups during the first week of post-weaning. When pups became adult animals the average dose of low-dose males and females had fallen to 15.4 and 17.4 mg aluminium/kg bw/d by week 9, decreasing to lower values during the rest of the study. Two control groups were additionally included receiving either sodium citrate solution (27.2 g/L; molar equivalent of the high-dose aluminium citrate) or water.

20 litters per dose group were kept after delivery. The litters were standardized to 4 pups/sex. One pup of each sex was assigned to one of four groups that were designed for the neurobehavioural testing on post natal days (PND) 23, 64, 120 and 364. Dams were exposed from gestational day 6 through lactation and then the offspring was exposed post-weaning until postnatal day 364.

Observations of the dams included water consumption, body weight, a functional observational battery, morbidity and mortality. Pups received one third to one half of the target doses of the dams for merely the complete treatment period, because lower water consumption was expected.

Pup observations included body weight twice weekly, fluid consumption weekly and a functional observational battery on all pups several times before weaning and twice weekly on the 1-year group until sacrifice. Performance in a T-maze and Morris water maze test as well as motor activity and startle response were examined at several times. At each sacrifice time (PNDs 23, 64, 120 and 364), half of the pups of each group were subjected for neurohistopathological examination. The other half was subjected to a regular necropsy followed by brain weight measurement, clinical chemistry, haematology, and collection of tissues and blood for measurement of aluminium and other metals. Furthermore female pups examined for starting of vaginal opening and male pups for preputial separation.

No mortality or severe clinical signs were observed for the dams. The majority of observations throughout the dosing groups consisted of mild dermatological lesions like alopecia or porphyrin staining. Furthermore diarrhea was observed in 8 animals of the high dose group. Thus, most observations were mild stress-related symptoms. Body weight of the treated dams was comparable to the control animals for the gestational and post-natal period. Water consumption was significantly higher in the low- and mid-dose groups; however no dose response was observed. No higher water consumption was observed in the high dose dams.

Aluminium-induced renal toxicity (hydronephrosis, urethral dilatation, obstruction and/or presence of calculi) was seen in pups of the high-dose group (300 mg Al/kg bw/d) and to a lesser extent in the mid-dose group (100 mg Al/kg bw/d) especially in males. Alterations of hind-limb and fore-limb grip strength (neuromuscular parameters) were observed in both males and females from 100 mg/kg bw. Those effects were partly considered secondary to body weight changes. No other major neurological pathology or neurobehavioural effects were observed. The effect on grip strength was stronger in younger animals. Thus, effects of exposure in utero and/or during lactation seem to be more important than exposure during the later stage. In fact these are the most sensitive time points relating to the developmental effects used as the critical end-point for the NOAEL derivation. Dams were treated with the target dose or higher during gestation and lactation period. Therefore, the lowering in the treatment dose noted in adult pups was not considered to have an impact on the study results. A delayed sexual maturation was observed in male and female offspring of the high dose group compared to lower doses. This effect was also observed in the sodium citrate group clearly suggesting a general ionic effect due to changes in water and/or food consumption.

Findings in the brain tissue observed during histopathological examination at study termination (364-day group) were seen both in treated and in control group animals. Thus this effect was not considered as treatment related and was likely considered to be due to aging. The levels of aluminium in tissue were generally regarded as dose related. The strongest association was observed regarding aluminium levels in bone.

Based on the Poirier et al. study (2011), the LOAEL was set at 100 mg/kg bw/d and the NOAEL at 30 mg/kg bw/d.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please refer to IUCLID section 13 for further information.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day

Based on:

other: recalculated for target substance

Sex:

male/female

Basis for effect level:

clinical signs

water consumption and compound intake

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

180 mg/kg bw/day

Based on:

other: recalculated for target substance

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

180 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproductive toxicity study is available for the target substance aluminium acetate. Therefore read across with the source substance aluminium citrate was performed. Please refer to IUCLID section 13 for the read across justification.

In the opinion of the world health organization (WHO) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) from 2011, one study performed with aluminium citrate was considered as valid and appropriate for other aluminium compounds and was thus also used to cover this endpoint for aluminium acetate. This was further confirmed by an opinion of scientific committee on consumer safety (SCCS) in 2014.

In a chronic developmental neurotoxicity study in accordance to OECD 426, aluminium citrate was administered to pregnant Sprague Dawley rats via drinking water. Treatment started on gestational day (GD) 6. The concentrations administered were aimed to deliver aluminium doses of 30, 100 and 300 mg/kg bw/d based on an expected daily water intake of 120 mL/kg bw/d. The treatment doses varied in the different phases due to changes in the water intake over time. During gestation the target dose was almost achieved at the low dose. During lactation the treatment dose for the dams was higher than 30 mg/kg bw/d (around 40 mg/kg bw/d). The mean dose was 40.2 and 43.5 mg (again higher than 30) for male and female pups during the first week of post-weaning. When pups became adult animals the average dose of low-dose males and females had fallen to 15.4 and 17.4 mg aluminium/kg bw/d by week 9, decreasing to lower values during the rest of the study. Two control groups were additionally included receiving either sodium citrate solution (27.2 g/L; molar equivalent of the high-dose aluminium citrate) or water.

20 litters per dose group were kept after delivery. The litters were standardized to 4 pups/sex. One pup of each sex was assigned to one of four groups that were designed for the neurobehavioural testing on post natal days (PND) 23, 64, 120 and 364. Dams were exposed from gestational day 6 through lactation and then the offspring was exposed post-weaning until postnatal day 364.

Observations of the dams included water consumption, body weight, a functional observational battery, morbidity and mortality. Pups received one third to one half of the target doses of the dams for merely the complete treatment period, because lower water consumption was expected.

Pup observations included body weight twice weekly, fluid consumption weekly and a functional observational battery on all pups several times before weaning and twice weekly on the 1-year group until sacrifice. Performance in a T-maze and Morris water maze test as well as motor activity and startle response were examined at several times. At each sacrifice time (PNDs 23, 64, 120 and 364), half of the pups of each group were subjected for neurohistopathological examination. The other half was subjected to a regular necropsy followed by brain weight measurement, clinical chemistry, haematology, and collection of tissues and blood for measurement of aluminium and other metals. Furthermore female pups examined for starting of vaginal opening and male pups for preputial separation.

No mortality or severe clinical signs were observed for the dams. The majority of observations throughout the dosing groups consisted of mild dermatological lesions like alopecia or porphyrin staining. Furthermore diarrhea was observed in 8 animals of the high dose group. Thus, most observations were mild stress-related symptoms. Body weight of the treated dams was comparable to the control animals for the gestational and post-natal period. Water consumption was significantly higher in the low- and mid-dose groups; however no dose response was observed. No higher water consumption was observed in the high dose dams.

Aluminium-induced renal toxicity (hydronephrosis, urethral dilatation, obstruction and/or presence of calculi) was seen in pups of the high-dose group (300 mg Al/kg bw/d) and to a lesser extent in the mid-dose group (100 mg Al/kg bw/d) especially in males.

Alterations of hind-limb and fore-limb grip strength (neuromuscular parameters) were observed in both males and females from 100 mg/kg bw. Those effects were partly considered secondary to body weight changes. No other major neurological pathology or neurobehavioural effects were observed. The effect on grip strength was stronger in younger animals. Thus, effects of exposure in utero and/or during lactation seem to be more important than exposure during the later stage. In fact these are the most sensitive time points relating to the developmental effects used as the critical end-point for the NOAEL derivation. Dams were treated with the target dose or higher during gestation and lactation period. Therefore, the lowering in the treatment dose noted in adult pups was not considered to have an impact on the study results.

A delayed sexual maturation was observed in male and female offspring of the high dose group compared to lower doses. This effect was also observed in the sodium citrate group clearly suggesting a general ionic effect due to changes in water and or food consumption.

Findings in the brain tissue observed during histopathological examination at study termination (364-day group) were seen both in treated and in control group animals. Thus this effect was not considered as treatment related and was likely considered to be due to aging. The levels of aluminium in tissue were generally regarded as dose related. The strongest association was observed regarding aluminium levels in bone.

Based on the Poirier et al. study (2011), the LOAEL was set at 100 mg/kg bw/d and the NOAEL at 30 mg/kg bw/d.

In accordance with REACH Annex XI section 1.2, third paragraph sufficient weight of evidence for the presence or absence of a particular dangerous property is available. Adequate and reliable documentation is provided for the chronic developmental neurotoxicity study according to OECD 426 and GLP. Further evaluation was performed by the WHO/JECFA. Considering the high bioavailability of aluminium citrate when compared to the other aluminium compounds, they concluded in their opinion that the NOAEL of 30 mg/kg bw/d can be considered as appropriate for other aluminium compounds. The SCCS came to the same conclusion in their opinion from 2014 and reported the NOAEL of 30 mg Al/kg bw/d as appropriate point of departure for systemic effects (cited in Final Opinion on tolerable intake of aluminium with regards to adapting the migration limits for aluminium in toys, 28 September 2017 by SCHEER (Scientific Committee on Health, Environmental and Emerging Risks)).

In conclusion, the study according to OECD 426 is regarded to provide sufficient information on repeated dose toxicity and thus further testing on vertebrate animals shall be omitted according to REACH Annex XI. This is further in line with REACH article 25 (1) that states that “In order to avoid animal testing, testing on vertebrate animals for the purposes of this Regulation shall be undertaken only as a last resort.”

Therefore, no OECD 422 study with the aluminium acetate was performed.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The NOAEL for reproductive toxicity was determined to be 180 mg/kg bw/d for the target substance. As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) No 2019/521.