Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 234-933-1 | CAS number: 12042-91-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
- Combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test (OECD 422, GLP, K, read-across, rel.1): NOAEL(systemic effects) = 1000 mg/kg bw/day ; NOAEL (reproduction/development) = 1000 mg/kg bw/day, ca. for dialuminium chloride pentahydroxide: NOAEL(systemic effects) = 300 mg/kg bw/day ; NOAEL (reproduction/development) = 300 mg/kg bw/day
- combined one-year developmental and chronic neurotoxicity study (OECD 426, GLP, K, read-across, rel.1):
NOAEL(maternal toxicity) = 300 mg (Al)/kg bw/day : NOAEL (pups) = 30 mg (Al)/kg bw/day, ca. for dialuminium chloride pentahydroxide: NOAEL(maternal toxicity) = 1000 mg/kg bw/day ; NOAEL (pups) = 100 mg/kg bw/day
Link to relevant study records
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Strengths of this study include the fact that it was conducted according to OECD TG 416,in according with GLP procedures, multiple dose levels were studied, both sexes were used, administered doses were analytically verified, a dose finding study was conducted and used doses were justified, stability of AAS in drinking solutions was detected and controlled. Limitations of this guideline study include a large gap (factor of 10) between the dose levels, urinalysis was not performed, lack of examinations/evaluation data on clinical signs of toxicity and absence of pH values for the AAS-containing drinking solution detract from the report. Few details were provided on the statistical methods used, blinding of observations, selection of the animals for the neurobehavioral tests, sperm parameters examination and the fact no measures of Al levels were conducted in the body fluids/organs. Possible irritant effects of AAS on the gastrointestinal tract mucosa of rats based on the results from the preliminary range-finding study could not be excluded.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- Deviations:
- yes
- Remarks:
- 10-fold interval between doses in this study, urinanalysis was not performed
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Atsugi Breeding Center, Charles river Laboratories Japan, Inc.
- Age at study initiation: (P) 5 wks
- Housing: animals were housed individually, except for the acclimation, mating and nursing periods, in metal-bracket-type cages with wire-mesh floor
- Diet: standard rat diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum, Al content in diet, analyzed by flame atomic absorption spectrometry for each lot of diet, ranged from 22 ppm to 29 ppm
- Water: deonized drinking water with (treatment group) or without (control group) dose concentrations, water given to controls contained < 5 µg Al/mL
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: drinking water
- Vehicle:
- other: deionized water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: fresh dosing solutions were prepared at least once every 7 days and the drinking water was replaced at least once every 5 days
- Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: until successful copulation occured or the mating period of 2 weeks had elapsed
- Proof of pregnancy: vaginal smears were examined daily for presence of sperm, and the presence of sperm in the vaginal smear and/or a vaginal plug were considered as evidence of successful mating, detection of sperm in the vaginal lavage was designated as day 0 of gestation
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged: from day 17 of gestation to day 21 after delivery, dams and litters were reared using wood chips as bedding - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- concentrations of AAS in drinking water were analyzed in the first and last preparations and once every 3 months, ans verified using high performance liquid chromatography (the quantitation limit -5 µg/mL)
- Duration of treatment / exposure:
- F0 males: 10 weeks prior to mating, during mating and up to termination after the parturition of paired females
F0 females: 10 weeks prior to mating, during mating and during lactation period until sacrifice after weaning of their pups (PND26)
F1: selected at PND21-25, exposure occured at the same doses and schedule as their parents - Frequency of treatment:
- 7 days/week
- Details on study schedule:
- - F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
- Age at mating of the mated animals in the study: 13-15 weeks - Remarks:
- Doses / Concentrations:
50, 500, 5000 ppm
Basis:
nominal in diet
for actual doses see table1 under any other information on materials and methods including tables - No. of animals per sex per dose:
- parental and F1: 24 males and females
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: the dose levels were selected based on the results of a dose-range finding study
- Rationale for animal assignment: by stratified random sampling based on body weight - Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all F0 and F1 parental rats were observed at least twice a day for general appearance and behavior. Dams were checked 3 times daily on days 21-25 of gestation.
BODY WEIGHT: Yes
- Time schedule for examinations: weekly through the study. For dams, body weight was recorded weekly until evidence of copulation was detected and then on gestational days 0, 7, 14 and 20 and days 0, 7, 14 and 21 of lactation.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined once a week and mean daily diet consumption calculated as g food/ day: Yes
Time schedule for examinations: weekly. For dams, food consumption was recorded through the exposure period, except during cohabitation.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice a week, through the exposure period, except during cohabitation. - Oestrous cyclicity (parental animals):
- Daily vaginal lavage samples were evaluated for each female for estrous cyclicity throughout the last 2 weeks before the cohabitation period and during cohabitation until evidence of copulation was detected. The presence of sperm in the vaginal smear and/ or a vaginal plug was considered as evidence of successful mating, and the day of successful mating was designated as day 0 of gestation.
- Sperm parameters (parental animals):
- Sperm parameters were determined in all F0 and F1 adult males on the day of sacrifice; The right testis was used to count testicular homogenization-resistant spermatid heads using a hemacytometer; The right epididymal cauda was weighed and used for sperm analysis (sperm number, sperm motility and morphology; Caudal sperm numbers were enumerated using a hemacytometer under a light microscope; For sperm motility (percentage of motile sperm and progressively motile sperm, swimming speed and pattern) were determined using a computer-assisted cell motion analyzer (TOX IVOS; Hamilton Thorne Bioscience, Beverly, MA, USA); Sperm morphology was evaluated for 200 sperm (stained with eosin and mounted on a slide glass) per male under a light microscope.
- Litter observations:
- STANDARDISATION OF LITTERS:
Performed on day 4 postpartum: Yes; maximum of 8 pups /litter (4/sex/litter), randomly selected.
PARAMETERS EXAMINED:
The following parameters were examined in all pups from F0 and F2 parents (F1 and F2 litters): number of pups, sex of pups, live birth, stillborn members per litter, and gross anomalies Clinical signs of toxicity (daily) and the body weight of live pups were measured on PND 0, 4, 7, 14 and 21.
GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.
OTHER:
Developmental landmarks:
- Pinna unfolding in all F1 and F2 live pups (for from PND 1 to PND 4); Eye opening beginning on PND12;
- The anogenital distance (AGD) was measured on PND 4 in all F1 and F2 pups using calipers, and the normalized value of AGD to body weight, AGD/cube root of the body weight ratio, was calculated (Gallavan et al., 1999);
- Incisor eruption beginning in one male and one female F1 and F2 pup selected from each dam were evaluated on PND 8 and eye opening beginning on PND 12, and continued until each pup achieved the criteria;
- The body weight of the respective F1 pups was recorded on the day the criteria were fulfilled.
Neuromotor performance:
- For the same F1 and F2 pups, surface righting reflex, negative geotaxis and mid-air righting reflex were assessed on PND 5, 8 and 18, respectively.
Sexual maturation:
- Preputial separation were observed daily for male in all F1 offspring selected as F1 parents beginning on PND 35 and
- Female vaginal opening were observed daily for female in all F1 offspring selected as F1 parents beginning on PND 25 until completion.
- The body weight of the respective F1 rats was recorded on the day of completion of these pubertal landmarks.
NEUROBEHAVIORAL EXAMINATIONS:
Locomotor activity:
- Spontaneous locomotor activity was measured in 10 male and 10 female F1 rats randomly selected from each group at 4weeks of age. A multi-channel activity monitoring system (SUPERMEX; Muromachi Kikai Co., Ltd., Tokyo, Japan) was employed.
Animals were placed individually in transparent polycarbonate cages [285(W)x 450(D) x 210(H) mm, CL-0108-1; CLEA Japan Inc., Tokyo, Japan], and spontaneous motor activity was measured using SUPERMEX (Muromachi Kikai Co., Ltd., Tokyo, Japan), which was placed above the cage to detect changes in heat across multiple zones of the cage with an infra-red sensor. Spontaneous motor activity was determined at 10-min intervals and for a total 60 min.
Spatial learning ability (T-maze test):
- Spatial learning ability was conducted using water-filled multiple T-maze test (Biel’s type) in 10 male and 10 female F1 rats selected from each group at 6 weeks of age.
The water temperature of the maze was maintained at 21.0–22° C. Each rat was allowed to swim three times in a straight channel on the day before the trial, and then tested in the maze with three trials per day for the following three consecutive days. The time required to reach the goal and the number of errors were recorded. To prevent the exhaustion, no animal was allowed to remain in the water for more than 3 min in any trial. - Postmortem examinations (parental animals):
- SACRIFICE
- F0 and F1 parental male animals: after a parturition of their paired females;
- Maternal animals: were evaluated for estrous cycle stage by examination of the vaginal smear after weaning of pups, and euthanized in the proestrus stage by exsanguination under ether anesthesia.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic, and abdominal viscera;
- The number of uterine implantation sites was recorded for each female;
- The testis and epididymis in males were prepared for microscopic examination and weighed;
- The brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididmydes, seminal vesicles (with coagulating glands and their fluids), ventral prostate, uterus and ovaries in males and females were weighed before fixation, fixed and underwent macroscopic examination; thyroid and seminal vesicles were weighed after fixation;
- The testis and epididymis were fixed with Bouin’s solution and preserved in 70% ethanol, and the other organs were stored in 10% neutral-buffered formalin.
- The number of primordial follicles in the right ovary was counted for 10 F1 females randomly selected from the control and highest dose groups. Every 20th section was mounted on a slide and stained with hematoxylin–eosin. About 40 sections per ovary were used to determine the primordial follicles.
HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluations were performed in F0 and F1 animals of the control and highest dose groups.
Of these animals, the testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina were embedded in paraffin ,sectioned, stained with hematoxylin–eosin and examined under a light microscope. - Postmortem examinations (offspring):
- SACRIFICE
Non-parental F1 weanlings and all F2 offspring were euthanized under ether anesthesia at PND 26 of age.
HISTOPATHOLOGY / ORGAN WEIGTHS
For one male and one female F1 and F2 weanlings selected from each dam:
- the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries were removed, weighted and prepared for microscopic examination;
-Since test substance-related organ weight changes were found in the thymus, liver and spleen weight and in the liver and spleen weight of females in the highest dose group in F1 and F2 generations, they were histopathologically examined for the randomly selected 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups. Paraffin sections were routinely prepared, stained with hematoxyllin-eosin and examined using a light microscope. - Statistics:
- Bartlett’s test for homogeneity of variances (p<0.05) was applied for homogeneity of distribution for parametric data (body weight, food and water consumption, length of estrous cycle and gestation, precoital interval, number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, parameters of behavioral tests, organ weight and sperm parameters);
For preweaning pups, body weight, AGD, viability, and age at completion of developmental landmarks were similarly analyzed using litter as experimental unit.
One way analysis of variance (p<0.10) was performed when homogeneity of distribution was established.
If a significant difference was detected, Dunnett’s test was conducted for comparisons between control and individual treatment groups (p<0.01 or 0.05).
Data without homogeneity were analyzed using Kruskal–Wallis rank sum test (p<0.10). If significant differences were found, Mann Whitney’s U test was conducted for comparison between control and each treatment group (p<0.01 or 0.05). Fisher’s exact test (p<0.01 or 0.05) was used to compare incidence of parental animals with clinical signs, and autopsy and histopathological findings, incidence of females with normal estrous cycles, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ratio and completion rate of negative geotaxis between AS and control group.
Wilcoxon rank sum test (p<0.01 or 0.05) was used to analyzed incidence of pups with clinical signs or autopsy findings per litter, completion rate of pinna unfolding in each litter, and success rate of surface and mid-air righting reflex.
Student’s t-test (p<0.01 or 0.05) was used to compare number of primordial follicles in control and highest dose groups because homogeneity of variance was indicated by F-test.
All of these statistical analyses were conducted using 5% level of probability as criterion for significance. - Reproductive indices:
- The following reproductive indices for each exposed group were calculated in F0 and F1 generation parental animals:
- Copulation index (%) for males and females(no. of animals with successful copulation/no. of animals paired)×100;
- Precoital interval(days);
- Fertility index(%) for males and females (no. of animals that impregnated a female or were pregnant/no. of animals with successful copulation)×100;
- Gestation index (%)(no. of females that delivered live pups/no. of pregnant females)×100;
- Gestation length (days);
- Delivery index(%)(no. of pups delivered/no. of implantations)×100;
- Estrous cycle in F0 and F1 females. - Offspring viability indices:
- For F1 and F2 offspring
Maternal indices:
No of litters;
No of pups delivered;
Sex ratio of pups total (no. of male pups/total no. of pups).
Viability index was calculated:
On PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered)×100;
On PND 4 (%) = (no. of live pups on PND 4/no. of live pups on PND 0)×100;
On PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) × 100.
Individual body weight:
Male and female individual weight during lactation on PND 0, 4, 7, 14 and 21. - Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- some effects in different live stages observed
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- some effects in different live stages observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance intake: details provided in table1
- Reproductive function: oestrous cycle:
- effects observed, treatment-related
- Description (incidence and severity):
- during premating period a few AAS treated animals had persistent diestrous (not adverse)
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- LOAEL
- Effect level:
- 36.3 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: - decreased liver, spleen and thymus weights in the F1 and F2 males and females - decreased body weight gain in the F1 and F2 males and females - delay in sexual maturation in F1 females
- Remarks on result:
- other: based on effects observed in F1 and F2 generations
- Dose descriptor:
- NOAEL
- Effect level:
- 5.41 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Remarks on result:
- other: based on effects observed in F1 and F2 generations
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- bodyweight for F1 males and females (5000 ppm) decreased significantly, for F2 (5000ppm) lower body weight but not significantly
- Sexual maturation:
- effects observed, treatment-related
- Description (incidence and severity):
- significantly delayed vaginal opening (F1 - 5000ppm)
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- see table3 + 4
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- LOAEL
- Generation:
- F1
- Effect level:
- 36.3 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: - decreased liver, spleen and thymus weights in the F1 and F2 males and females - decreased body weight gain in the F1 and F2 males and females - delay in sexual maturation in F1 females
- Remarks on result:
- other: based on effects observed in F1 and F2 generations
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 5.41 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: based on effects observed in F1 and F2 generations
- Dose descriptor:
- LOAEL
- Generation:
- F2
- Effect level:
- 36.3 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: - decreased liver, spleen and thymus weights in the F1 and F2 males and females - decreased body weight gain in the F1 and F2 males and females - delay in sexual maturation in F1 females
- Remarks on result:
- other: based on effects observed in F1 and F2 generations
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 5.41 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Remarks on result:
- other: based on effects observed in F1 and F2 generations
- Reproductive effects observed:
- not specified
- Conclusions:
- The major findings reported by the authors were decreased body weight in preweaning animals, delayed maturation of the female offspring, and decreased organ weight in the offspring. However, because the effects could be related to decrease fluid consumption and limited nursing ability of dams, the utility of this study for risk assessment is limited.
Limited examination of the internal and external abnormalities, morphological variations and malformations precludes rigorous assessment of the teratogenic potential of the AAS test compound. As urinanalysis was not performed, possible adverse effects of prolonged AAS ingestion on kidney function cannot be assessed. - Executive summary:
Hirata-Koizumi et al. (2011b) investigated the potential reproductive toxicity of aluminium ammonium sulfate (CAS#: 7784-25-0 (anhydrous)) CAS#: 7784-26-1 (dodecahydrate)] in a GLP and OECD TG 416 -compliant 2 generation reproductive toxicity study.
Aluminium ammonium sulphate (AAS) was dissolved in deionized water at 0, 50, 500 or 5000 mg/L. The Al concentration in the deionized water was < 5 µg/mL and the Al content of the diet was 22-29 mg/kg Groups of 24 male and 24 female Crl:CD (SD) rats (F0 generation) were given AAS in drinking water from 5 weeks of age for 10 weeks prior to mating, during mating and gestation, when the parental males were culled, and for the females through weaning. Litters were normalized to 8 pups on PND 4. At weaning, 24 males and 24 females were selected to serve as the F1 generation and they were given AAS in drinking water for 10 weeks prior to mating, during mating and gestation, and for the females through weaning, as for the F0 generation. Exposure of the F1 weanlings occurred at the same concentrations as those of their parents.
Spontaneous locomotor activity was assessed at 4 weeks of age in 10 male and 10 female randomly selected F1 pups per group. Rats were placed in transparent polycarbonate cages and observed using an infrared sensor. Observations were made at “10 minute periods and for a total of 60 minutes.” At 6 weeks of age a water-filled multiple T-maze was used to assess the spatial learning abilities of 10 male and 10 female F1 pups from each treatment group (selected randomly). Habituation of swimming ability was conducted by allowing the animals to swim three times in a straight channel the day before the trial. Testing was done in blocks of three trials on three consecutive days. Rats were restricted to 3 min in the water to prevent physical exhaustion, but whether any rats were excluded on the basis of their performance was not reported. The parameters recorded to assess performance included time from entry into the water, elapsed time to traverse the straight channel, reaching the “goal ramp” and the numbers of errors.
Drinking water consumption was reduced at all concentrations compared to that of the concurrent controls. These reductions were clearly concentration-dependent and the reduction was significant at 500 and 5000 ppm in males and females of the F0 and F1 generations. These reductions were significant at 50 ppm in the F0 males and at some intervals during AAS exposure of the F0 and F1 females.
A transient decrease in food consumption was observed in the 500 and 5000 ppm groups and in body weight in the 5000 ppm group. One F1 male in the 500 ppm group died, but that death was not considered treatment-related.
There were no significant effects of AAS consumption on the oestrus cycle. The authors reported no differences for copulation, fertility index, gestation index, precoital interval, gestation length, number of implantations, live pups delivered or delivery index, sex ratios of pups or viability during the preweaning period in females compared to the control. There were no significant differences between control and AAS-treated groups regarding the numbers of testis and cauda epididymal sperm, percentage of motile and progressively motile sperm, sperm swimming patterns and speed or the numbers of morphologically-abnormal sperm. Moreover, there were no significant differences in the numbers of primordial follicles in the F1 ovaries between animals given 5000 ppm AAS and those consuming deionized water.
In the F1 and F2 pups, there were no treatment-related differences in numbers of offspring with congenital malformations, sex-ratio or viability on PND 0, 4 or 21. Reduced body weights were reported in the F1 male and female pups at 5000 ppm, but not in lower dose groups. The F1 male pups had a significantly lower body weight on PND 21, F1 female pups on PND 14 and 21, and F2 male and female pups on the PND 26. In female F1 pups, vaginal opening was delayed significantly among those whose mothers consumed 5000 ppm AAS (mean ± S.D: 32.3 ± 1.8 days vs. 30.2 ± 2.1 days in control), but their body weights were not significantly different from those of the concurrent control at the time of vaginal opening.
Absolute weights of testes and epididymis of the F1 and F2 male pups at 5000 ppm were lower than control. Absolute weights of the uterus were significantly lower in the F1 female pups, and absolute weights of the ovary and uterus were significantly lower in the F2 females. Histopathological examination revealed no treatment-related changes in the reproductive organs. The authors considered these findings secondary to the decreased body weights and attributed the reductions in growth and development of the offspring “to the astringent taste of AAS which would decrease the palatability of drinking water in the AAS-treated groups”.
Spontaneous locomotor activity was no different among F1 males from dams given AAS in drinking water and those whose mothers consumed deionized water alone. There was some variation in activity among the F1 females.
The results presented by 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, Hirata-Koizumi et al. (2011b) 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 preweaning 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 (Table 1, p.6). The reported NOAEL is 500 mg AAS/L which corresponds to 5.35 mg Al/kg bw per day.
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008 - 2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Comparable to guideline study with acceptable restrictions. (Spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured).
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
- Deviations:
- yes
- Remarks:
- spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Japanese guidelines for the "designation of food additives and for the revision of standards for the use of food additives".
- Deviations:
- not applicable
- GLP compliance:
- yes
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: (P) 5 wks; (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing:
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):
IN-LIFE DATES: From: To: - Route of administration:
- oral: drinking water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared at least every 6 days and kept under cool conditions until serving and drinking solutions were replaced at least once every 4 days
- Details on mating procedure:
- - M/F ratio per cage: 1:1 from the same treatment group
- Length of cohabitation: until successful copulation occured or the mating period of 2 weeks had elapsed
- Proof of pregnancy: during mating period vaginal smears were examined daily for the presence of sperm, presence of sperm in vaginal smears/or vaginal plug were considered as evidence of successful mating (day0 of gestation)
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: after day17 of gestation to day21 after delivery, wire-mesh floors of cages was replaced with stainless steel tray and individual dams or litters were reared using wood chipsas bedding - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- during the study the, the concentrations of AS in drinking water were analyzed in the first and last preparations and once every 3 months, and verified using high performance liquid chromatography (quantitation limit 5 µg/mL)
- Duration of treatment / exposure:
- F0 males: 10 weeks prior to mating, during mating and up to parturition of the paired females
F0 females: 10 weeks prior to mating, during mating and during lactation until sacrifice after weaning of their pups (PND26)
F1: selected on PND 21-25 (designated as day0 of dosing) and were treated in same way as F0 males and females - Frequency of treatment:
- animals were dosed up to the day prior to necropsy
- Details on study schedule:
- - F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
- Age at mating of the mated animals in the study: 13 weeks (F1), 15 weeks (F0) - Dose / conc.:
- 120 ppm (nominal)
- Remarks:
- Doses / Concentrations: 120, 600, 3000 ppm Basis: nominal in diet for actual doses received see table1 under any other information on materials and methods including tables
- Dose / conc.:
- 600 ppm (nominal)
- Remarks:
- Doses / Concentrations: 120, 600, 3000 ppm Basis: nominal in diet for actual doses received see table1 under any other information on materials and methods including tables
- Dose / conc.:
- 3 000 ppm (nominal)
- Remarks:
- Doses / Concentrations:
120, 600, 3000 ppm
Basis:
nominal in diet
for actual doses recieved see table1 under any other information on materials and methods including tables - No. of animals per sex per dose:
- 24 animals per sex and group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: the dose levels were selected based on the results of a preliminary range-finding study
- Rationale for animal assignment: by stratified random sampling based on body weight - Positive control:
- not required
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day
BODY WEIGHT: Yes
- Time schedule for examinations: weekly, For dams body weight was recorded on gestational days 0, 7, 14 and 20 and days 0, 7, 14 and 21 of lactation (and additional day 4 of lactation for body weight)
FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Time schedule for examinations: weekly. for dams, food consumption was recorded on gestational days 0, 7, 14 and 20 of gestation and days 0, 7, 14 and 20 of lactation (and additional day 4 of lactation for body weight)
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice a week and on days 0, 7, 14 and 20 of gestation and days 0, 4, 7, 11, 14, 17, 19 and 21 of lactation - Oestrous cyclicity (parental animals):
- Daily vaginal lavage samples were evaluated for each female for estrous cyclicity throughout the last 2 weeks of the premating period and during cohabitation until evidence of copulation was detected. Females with repeated 4-6 day estrous cycles were considered as having normal estrous cycles.
- Sperm parameters (parental animals):
- Sperm parameters were determined in all F0 and F1 adult males on day of sacrifice; right testis was used to count testicular homogenization-resistant spermatid heads; the right epididymal cauda was weighed and used for sperm analysis.
For sperm motility, the percentage of motile sperm and progressively motile sperm and the swimming speed and pattern were determined using a somputer-assissted cell motion analyzer (TOX IVOS). After recording sperm motion, the cauda epididymalfluid was diluted and the sperm were enumerated with a hematocytometer und a light microscope. Sperm count per gram of epididymal tissue was obtained by dividing the toal count by the gram weight of the caudal epididymis.
Sperm orphology was studied by stainign sperm with eosin and mount it on glass slides. 200 sperm in each sample were examined under light microscope and percentage of morphologically abnormal sperm was calculated. - 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, no adjustment was made for litters of fewer than eight pups); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
Number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities. clinical signs of toxicity (daily) and the body weight of live pups were measured on PND 0, 4, 7, 14 and 21.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
OTHER:
- developmental landmarks: Pinna unfolding in all F1 and F2 pups (PND1 - PND4); anogenital distance (AGD) measured on PND4 in all F1 and F2 pups; incisor eruption for one male and one female F1 and F2 pup selected from each liter were evaluated on PND8 and eye opening on PND12 until each pup fulfilled criteria
- neuromotor performances: surface righting reflex, negative geotaxis and midair righting reflex were assessed on PND5, 8 and 18 for one male and female F1 and F2 pup selected from litter
- neurobehavioral examinations:
locomotor activity - 10 male and 10 female F1 rats randomly selected from each group at 4 weeks of age (multi-channel activity monitoring system used)
T-maze test - water-filled multiple T-maze test was conducted in 10 male and 10 female F1 rats selected from each group at 6 weeks of age - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced (after a parturition of their paired females).
- Maternal animals: All surviving animals, after the last litter of each generation was weaned, on PND26.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
The number of uterine implantation sites was recorded for each dam. the testis and epididymis were prepared for microscopic examination and weighed. The brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididymis, seminal vesicles (with coagulating gland and their fluids), ventral prostate, uterus and ovaries in males and females were weighed before fixation, fixed and underwent macroscopic examination. The thyroid and seminal vesicles were weighed after fixation. In 10 F1 females, randomly selected from the control and highest dose group, the number of primordial follicles was counted in about 40 sections per ovary.
HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathologic evaluations were performed:
- in all animals of the control and the highest dose group
- in females with abnormal estrous cycle, abnormal delivery or total dead pups
- in males and females without evdence of copulation or insemination
- in all animals with grossly abnormal reproductive organs
- testes and epididymis were fixed in Bouin's solution and preserved in 70% ethanol; all other organs were fixed in 10% neutral bufferes formalin.
Testes, epididymis, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina were sectioned, stained with hematoxylin-eosin and examined under a light microscope. When treatment-related changes were found in the highest dose group, the same tissue from the next lower doese group then were examined. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 26 days of age.
- all pups found dead before weaning were necropsied immediately, following the adjustment of litter size on PND4, culled pups were sacrifized by carbon monoxide and subjected to gross external and internal examination
HISTOPATHOLOGY / ORGAN WEIGTHS
for one male and one female F1 and F2 weanlings selected from each dam:
- the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries were removed and prepared for microscopic examination and weighed
- Since test substnace-related organ weigth changes were found in liver and spleen of highest dose group in F1 and F2 generations, these tissues were histopathologically examined for 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups
- If treatment-related histopathological changes were observed in the highest dose group, the same tissue in the next lower dose group was examined as well. - Statistics:
- Bartlett's test: was applied for homogeneity of distribution for parametric data (body weight, food and water consumption, length of estrous cycle and gestation, precoital interval, the number of implantations and pups born, delivery index, reflex response time, age atsexual maturation, behavioral test parameters, organ weight and sperm parameters);
For preweaning pups, body weight, AGD, viability, and age at the completion of developmental landmarks were similarly analyzed using the litter as the experimental unit.
One way analysis of variance was performed when the homogeneity of distribution was established.
If a significant difference was detected, Dunnett's test was conducted for comparison between control and individual treatment groups.
Data without homogeneity were analyzed using the Kruskal-Wallis rank sum test. If significant differences were found, the Mann Whitneys's U test was conducted fr comparison between the control and each dose group.
Fisher exact test was used to compare the incidence of parental animals with clinical signs, and autopsy and histopathological findings, the incidence of females with normal estrous cycle, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ration and completion rate of negative geotaxis between the AS and control group.
The Wilcoxon rank sum test was used to analyze the incidence of pups with clinical signs and necroscopy findings per litter, the completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex.
Student's T-test was used to compare the number of primordial follicles in the control and highest dose group because the homogeneity of variance was indicated by the F-test.
All of these statistical analysis were conducted using the 5% level of probability as the criterion for significance. - Reproductive indices:
- - Copulation index (for males and females) (%): (no. of animals with successful copulation/no. of animals paired) x 100
- precoital interval (days)
- fertility index (for males and females) (%): (no. of males that impregnated a female or no. of pregnant/no. of animals with successful copulation) x 100
- Gestation index (%): (no. of females that delivered live pups/no. of pregnant females) x 100
- Gestation length (days)
- Delivery index (%): (no. of pups delivered/no. of implantations) x 100
- Estrous cycle in F0 and F1 females - Offspring viability indices:
- For F1 and F2 offspring:
Maternal indices; no. of litters; no. of pups delivered; sex of all pups; sex ration of pups total (no. of male pups/total no. of pups)
Viability index calculated:
on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) x 100
on PND 4 (%) = (no. of live pups on PND 4/ no. of live pups on PND 0) x 100
on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) x 100 - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- no significant difference seen between control and AS treated groups in incidence of clinical signs; 120 ppm: one F1 female died (non adverse); 600 ppm: one F0 female died (non adverse); 3000 ppm: one F1 femlae died (non adverse)
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance intake: administered in drinking water
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 31.2 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: Decreased body weight gain, decreased food consumption
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 8.06 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: Not reported
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 53 mg/kg bw/day
- Based on:
- other: For Aluminium sulphate (convert using 342.1 g/mol)
- Sex:
- female
- Basis for effect level:
- other: Reproductive Toxicity, rat, daily dose 6 weeks, maternal, female, gestational length
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Since the dosing solution containing AS was
pH 3.57–4.20, the acidity would decrease the palatability of drinking water in AS-treated groups. Decreased water consumption was
associated with decreased food consumption by F0 and F1 males
and females in the 600 and 3000 ppm groups and decreased body
weight in F0 male and females in the 3000 ppm group. Since waterdeprived animals typically reduce their levels of feed consumption
and consequently lower their body weight - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Since the dosing solution containing AS was
pH 3.57–4.20, the acidity would decrease the palatability of drinking water in AS-treated groups. Decreased water consumption was
associated with decreased food consumption by F0 and F1 males
and females in the 600 and 3000 ppm groups and decreased body
weight in F0 male and females in the 3000 ppm group. Since waterdeprived animals typically reduce their levels of feed consumption
and consequently lower their body weight - Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- Since the dosing solution containing AS was
pH 3.57–4.20, the acidity would decrease the palatability of drinking water in AS-treated groups. Decreased water consumption was
associated with decreased food consumption by F0 and F1 males
and females in the 600 and 3000 ppm groups and decreased body
weight in F0 male and females in the 3000 ppm group. Since water deprived animals typically reduce their levels of feed consumption
and consequently lower their body weight. - Reproductive function: oestrous cycle:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 53 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- For Aluminium sulphate (convert using 342.1 g/mol)
- Sex:
- male/female
- Basis for effect level:
- other: Not reported
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 8.06 ca. mg/kg bw/day (actual dose received)
- Based on:
- other:
- Sex:
- male/female
- Basis for effect level:
- water consumption and compound intake
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Two F1 pups showed malformations (non adverse), no malformed F2 pups were observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 3000 ppm: F1 males and females - body weights were significantly lower on PND21; F2 females - body weights were significantly lower than controls on PND21; males - no significant differences
- Sexual maturation:
- effects observed, treatment-related
- Description (incidence and severity):
- 3000 ppm: vaginal opening was significantly delayed (F1)
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- 3000 ppm: absolute and relative liver weights and absolute spleen weight, absolute weight of thymus, kidney, testes and epididymides decreased; absolute weight of uterus decreased at 600 ppm; relative brain weight decreased
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Key result
- Dose descriptor:
- LOAEL
- Generation:
- F1
- Effect level:
- 31.2 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: decreased preweaning body weight gain in the F1 and F2 males and females; decreased absolute and relative liver and spleen weights in the F1 and F2 males and females; delayed vaginal opening in F1 females
- Key result
- Dose descriptor:
- LOAEL
- Generation:
- F2
- Effect level:
- 31.2 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: decreased preweaning body weight gain in the F1 and F2 males and females; decreased absolute and relative liver and spleen weights in the F1 and F2 males and females; delayed vaginal opening in F1 females
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 8.06 mg/kg bw/day
- Based on:
- test mat.
- Remarks:
- Al
- Sex:
- male/female
- Basis for effect level:
- other: Not reported
- Key result
- Reproductive effects observed:
- not specified
- Treatment related:
- not specified
- Conclusions:
- Interpretation of the results is difficult due to the clear effect of AS treatment on fluid consumption. Addition of the test substance 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 levels, the F0 and F1 females also decreased their food consumption relative to the controls during week 3 of lactation. As a result, due to decreased drinking water consumption and decreased food consumption of F0 and F1 dams during the later stages of lactation, it is not possible to conclude with certainty whether the observations reported were associated with Al or represent secondary effects due to maternal dehydration and reduced nursing that may have influenced pup weight on PND 21. Because the effects reported could be related to decreased maternal fluid consumption, the utility of this study for risk assessment is limited.
- Executive summary:
In an OECD TG 416 and GLP compliant studies, aluminium sulfate Al2(SO4)3 (AS) was administered by a relevant oral route with drinking water to Crl: CD(SD) rats at multiple dose levels (120, 600 and 3000 ppm) before mating, during mating, gestation and lactation period in the two generation reproductive toxicity study. Twenty-four animals per sex and group (F0 and F1 generation) were given AS 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 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 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 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 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 and the authors suggested their NOAEL was conservative.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 September 2006 - 03 November 2006
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Study was conducted according to OECD guideline 422 and under GLP conditions.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- 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 or test system 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
- Vehicle:
- water
- Remarks:
- (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
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+ - 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 ; littering and post-mortem examination
- 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
- Dose / conc.:
- 40 mg/kg bw/day (actual dose received)
- Remarks:
- actual ingested
- Dose / conc.:
- 200 mg/kg bw/day (actual dose received)
- Remarks:
- actual ingested
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- actual ingested
- Dose / conc.:
- 3.6 mg/kg bw/day (actual dose received)
- Remarks:
- actual ingested Aluminium
- Dose / conc.:
- 18 mg/kg bw/day (actual dose received)
- Remarks:
- actual ingested Aluminium
- Dose / conc.:
- 90 mg/kg bw/day (actual dose received)
- Remarks:
- actual ingested Aluminium
- 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:
Observations: Mortality / viability at least twice daily
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):
- No clinical signs of toxicity noted.
Incidental findings that were noted consisted of salivation and alopecia of various body parts. These findings are commonly noted in rats of this age and strain which are housed and treated under the
conditions in this study.
No clinical signs were noted in control males, males at 40 mg/kg bw/day and in both sexes at 200 mg/kg bw/day. - Mortality:
- no mortality observed
- Description (incidence):
- One female sacrificed in extremis due to parturition difficulties on day 22 p.c.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Slightly lower mean bw for females at 1000 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 - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Mean food consumption of females at 1000 mg/kg bw/d slightly lower than controls. Otherwise no deviations compared to controls
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Minor, but statistically significant lowering of MCHC in M and F of 1000 mg/kg bw/g group
Minor, but statistically significant higher plateled count in males at 1000 mg/kg bw - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Males at 1000 mg/kg bw/d: statistically significant
-lower ALP activity
-lower albumin levels
-higher Potassium levels
-higher inorganic phospate levels
No deviations in females or in males at 40 & 200 mg/kg/d - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- All parameters (hearing ability, pupillary reflex, static righting reflex and grip strength) were normal in all animals.
The variation in motor activity did not indicate a relation with treatment - Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Mild to moderate subacute inflammation of the glandualr mucosa and minimal to moderate superficial easinophilic spheroids in all examined animals of both sexes at 1000 mg/kg bw/d
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- effects observed, treatment-related
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- % pregnant per dose level:see under details on results
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- 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:
- 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
Litter weights: No difference between exposed and controls - Sexual maturation:
- not specified
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- The slightly higher brain weights of female pups of the 1000mg/kg bw/day group were of a minor nature and considered not to be indicative of an adverse effect on pup development. No statistically significant difference towards control pups was noted when brain weights were corrected for body weights.
- 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 - Histopathological findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No behavioural abnormalities in pups observed
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- 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:
- Al 3+
- Sex:
- male/female
- Basis for effect level:
- other: No abnormalities at any dose level
- Reproductive effects observed:
- not specified
- Conclusions:
- 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) showed no maternal toxicity at any dose and no reproductive, breeding or developmental toxicity at any dose.
Therefore, a NOAEL for maternal local and systemic toxicity of 1000 mg/kg bw/d for aluminium chloride basic (equivalent to 90 mg/kg bw/d Al3+) was established. For reproductive and developmental toxicity the NOAEL was 1000 mg/kg bw /d for aluminium chloride basic (equivalent to 90 mg/kg bw/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 (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+)
The NOAEL for paternal systemic toxicity is 1000 mg/kg bw/d (equivalent to 90 mg/kg bw/d Al3+).
The NOAEL for maternal local and systemic toxicity is 1000 mg/kg bw/d (equivalent to 90 mg/kg bw/d Al3+).
The NOAEL for reproductive and developmental toxicity is 1000 mg/kg bw /d (equivalent to 90 mg/kg bw/d Al3+)
- Endpoint:
- one-generation reproductive toxicity
- Remarks:
- Developmental neurotoxicity and chronic toxicity study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008-2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: OECD 426 and OECD 452
- Deviations:
- yes
- Remarks:
- : food consumption was not studied; exposure during in utero (GD 6-21) and weaning period (post-natal day (PND) 1-21), but the exposure of the rats to Al citrate continued beyond this period, until 12 months of age in one cohort
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Canada Inc.
- Age at study initiation: 14 - 16 weeks at breeding
- Weight 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. - 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. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Verification of Al concentrations in the formulations and dosing solutions
The formulations and dosing solutions were prepared based on the Al content specified in the supplier’s Certificate of Analysis. Samples of at least 5 mL of each dose level of the dosing solution and also for the sodium citrate reference solution were stored and transported (overnight; ambient temperatures) then analyzed for aluminium content by ICPMS. Samples were collected from the first formulation, then from each week’s formulation for 4 weeks, then at 4 week intervals and, at the last dose preparation, until the end of the study.
The analyses showed that the dosing solutions prepared from the third lot of Al citrate had unexpectedly low Al concentrations, about 25% below target. The amount of Al citrate was thus increased to compensate. The Certificate of Analysis from the supplier gave a nominal concentration of 8.7% Al for this lot of the test item. The lower than specified Al levels (6.6% by analysis) were later confirmed by the supplier.
The Al concentrations in the dosing solutions differed from target by -30% to +39% throughout the study (Appendix B, Table B26. Dose Verification).
The stability and homogeneity of the dosing solutions under test conditions were determined in a separate study (Appendix F: Evaluation of the Stability and Homogeneity of Aluminium Citrate in Sodium Citrate and Aluminium Citrate Dosing Solutions by ICP-MS; ALB206-070144-5120). The results indicated that aluminium concentrations (at 2.5 g/L Al-citrate or endogenous Al levels in 27.2 g/L sodium citrate) remained stable and well-mixed in aqueous solution in a feeding bottle at room temperature for a 21 day period.
Aluminium Levels in the Diet and Vehicle
Samples of the different diets were analysed for aluminium, iron, manganese, copper, and zinc. For the enriched Purina AIN-93G, one sample was collected prior to the study and another was collected 6 weeks after the experimental starting date. One sample of Purina AIN-93M was taken prior to the switch in diets and another 6 weeks later. When new lots of the maintenance diet were received, they were tested before entering the study and again 6 weeks after being introduced.
Levels of aluminium in the diets were 6-9 ppm (6-9 µg/kg diet) over the study.
Levels of aluminium in the Nanopure water ranged from <1 – 160 ppb or 1 ug Al/L- 160 ug Al/L
Aluminium levels in the Reference Item
Aluminium levels were also determined similarly in the sodium citrate solutions. Dose verification analyses showed levels from 40-249 µg Al/L (with 6 of 19 measurements ≥100 µg Al/L; see Appendix B, Table B26).
All analyses were appropriately blinded. - Duration of treatment / exposure:
- On gestational day 6, the test item was administered to groups of pregnant animals during gestation, lactation, and to offspring during post-weaning, through to post-natal day 364 for cohort 4.
Dams
GD 6 to PND 21.
Pups (males and females)
PND 22 to PND 364.
Cohort 1 – GD 6-21, PND 1-22
Cohort 2 – GD 6-21, PND 1-64
Cohort 3 – GD 6-21, PND 1-120
Cohort 4 – GD 6-21, PND 1-364 - Frequency of treatment:
- ad libitum (daily, 7 days per week.)
- Details on study schedule:
- No data
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- Group A - low dose group (mg Al/kg bw/day)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Group D - mid dose group (mg Al/kg bw/day)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Group E - high dose group (mg Al/kg bw/day)
- Dose / conc.:
- 0 other: control I
- Remarks:
- Group C - Distilled water
- Dose / conc.:
- 27.2 other: g/L; control II
- Remarks:
- Group B - Na citrate group (27.2 g citrate/L)
- 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. - Positive control:
- No positive control group exhibiting central nervous system pathology and peripheral nervous system pathology available.
- Parental animals: Observations and 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. - Oestrous cyclicity (parental animals):
- No data.
- Sperm parameters (parental animals):
- No data.
- Litter observations:
- Pups
Body weights
Schedule: PND 1, 4 (prior to assignment to cohorts), 8, 11, 15, 17, 22, 29, and biweekly thereafter (with the exception that a 13-day interval was used between PND 43 and 56), and immediately prior to sacrifice.
Water consumption
Schedule: Cohorts 2 to 4 (Days 64, 120 and 364) on PND 22 and weekly thereafter until just prior to sacrifice.
Note: The pups in the Day 23 (Cohort 1, pre-weaning cohort) cohort received their own water bottles for one day after weaning and before sacrifice, but water consumption was not measured in these animals.
Developmental landmarks
Female pups were monitored for vaginal opening starting on PND 26.
Male pups were monitored for preputial separation starting on PND 35.
Blood collection
Selection of pups: Ten males and ten female pups from each treatment group were randomly selected for blood collection.
Methods: Terminal blood samples were taken from anesthetized animals on the day of scheduled sacrifice, prior to euthanasia. Venipuncture of the abdominal vena cava was used with the exception of Cohort 1(Day 23) animals which required cardiac puncture due to the small size of the rats.
Blood analysis - Clinical chemistry
In serum, alanine aminotransferase , albumin ,albumin/globulin ratio, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatine kinase, creatinine, globulin, glucose, sorbitol dehydrogenase, phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea nitrogen were measured.
Haematology parameters
The following parameters were evaluated on an Abbott Cell-Dyn® 3700 CS using Abbott reagents:
- Red Blood Cell count and morphology
- White Blood Cell count
- Differentiation of Granulocytic and Agranulocytic White Blood Cell
- Haematocrit
- Haemoglobin
- Mean Cell Haemoglobin
- Mean Cell Volume
- Mean Cell Haemoglobin Concentration
- Platelet count.
Coagulation panel
Prothrombin time (PT) and partial thromboplastin time (PTT) were assessed using a Coagamate® XM with Somagen reagents. .
Aluminium levels in blood
Blood samples (200 μL) of animals undergoing normal necropsy were taken into polypropylene containers and sent to the test site for analysis by ICP-MS.
Exclusion of samples
Samples with blood clots with largest dimension >2 mm were not run for haematology.
Samples obtained by cardiac puncture were included in analyses as long as sample quality was adequate, recognizing that samples collected by this method may contain artifactually high levels of creatinine kinase and aspartate aminotransferase.
Most haemolysed samples of sufficient quality were included in clinical chemistry analyses. For all assays with the exception of aspartate aminotransferase, samples that were excluded exceeded the maximum allowable haemolysis index specified by the manufacturer of the reagents. - Postmortem examinations (parental animals):
- No data.
- Postmortem examinations (offspring):
- Necropsy of Animals Undergoing Terminal Blood Collection/Analysis of Metal Levels in Tissues
Half of the animals scheduled to be sacrificed at the end of each observation period (10 males and 10 females per treatment group planned) were euthanized by exsanguinations under isoflurane anaesthesia and underwent a necropsy supervised by a Board-Certified Veterinary Pathologist. Animals that were found dead during the study also underwent a necropsy.
Brain weight
The brains of these animals were dissected and weighed. Brain weights were not recorded for rats that were found dead or were euthanized prior to the end of the study, including the culls.
Liver and left kidney tissues were collected and stored in neutral buffered formalin (10%). Right kidney tissue was collected and frozen at -10ºC.
Necropsy of Animals Undergoing Perfusion Fixation
Half of the animals scheduled to be sacrificed at the end of each observation period (10 males and 10 females per treatment group planned) were euthanized by perfusion fixation and underwent a necropsy under the supervision of a Board Certified Veterinary Pathologist.
At the rest of the sacrifice dates (postnatal Days 64, 120 and 364), the animals assigned to perfusion fixation were littermates of the animals assigned for perfusion fixation from the Day 23 cohort.
At the discretion of the study director, animals from the perfusion group found dead or euthanized prior to the end of the study underwent a regular necropsy conducted by or under the supervision of a veterinarian.
Histology (Tissues Undergoing Perfusion Fixation)
The following tissues (collected into 10% neutral buffered formalin)
- brain regions (5 locations - cerebrum at the optic chiasm, cerebrum at the base of the posterior hypothalamus, mid-cerebellum and medulla oblongata, pons at the” middle of its protrusion”, and the cranial cervical cord);
- spinal Cord (cervical and thoracic over at least 3 vertebrae each (at two levels));
- lumbar spinal roots (cauda equina);
- dorsal root ganglia;
- sciatic nerve (one proximal and one distal section; one transverse and one longitudinal section at each level); and
- skeletal muscle (gastrocnemius-soleus muscle)
were examined for cellular alterations and other changes, with a particular “emphasis on structural changes indicative of developmental insult”.
Examples of particular changes of interest were provided:
1. Gross changes in the size or shape of brain regions such as alterations in the size of the cerebral hemispheres or the normal pattern of foliation of the cerebellum.
2. The death of neuronal precursors, abnormal proliferation, or abnormal migration as indicated by pyknotic cells, or ectopic neurons, or gross alterations in regions with active proliferative and migratory zones, alterations in transient developmental structures (e.g., the external germinal zone of the cerebellum).
3. Abnormal differentiation, while more apparent with special stains, may also be indicated by shrunken and malformed cell bodies.
4. Evidence of hydrocephalus, in particular enlargement of the ventricles, stenosis of the cerebral aqueduct and general thinning of the cerebral hemispheres.
Slides were also examined for more typical cellular alterations such as neuronal vacuolation, degeneration necrosis) \and more typical tissue changes such as (astrocytic proliferation, leukocytic infiltration and cystic formation).
Slides were prepared according to GLP consistent with a SOP and the study protocol. Wet tissue was processed, embedded in glycol methylmethacrylate (GMA), sectioned and stained with hematoxylin and eosin (H&E). Tissues were sectioned according to Registry of Industrial Toxicology Animal data guidelines. In appendix I of the final report it is stated that quality checks of the tissue processing were conducted to ensure that it had been appropriate. All slides were then sent for examination by the study veterinary pathologist who was blind to the treatment group. - Statistics:
- See "any other information on materials and methods incl. tables"
- Reproductive indices:
- gestational length
- Offspring viability indices:
- mortality and morbidity
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 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. - Mortality:
- no mortality observed
- Description (incidence):
- 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 and weight changes:
- no effects observed
- Description (incidence and severity):
- 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.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Water consumption and compound intake (if drinking water study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- 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. - Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- 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. - Organ weight findings including organ / body weight ratios:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- Gestation Length:
There were no statistically significant differences in gestational length between the different treatment groups. - Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- not specified
- Dose descriptor:
- NOAEL
- Remarks:
- Maternity toxicity
- Effect level:
- 3 225 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no treatment-related adverse effects
- Dose descriptor:
- NOAEL
- Remarks:
- Maternity toxicity
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- element
- Remarks:
- Al 3+
- Sex:
- female
- Basis for effect level:
- other: no treatment-related adverse effects
- Dose descriptor:
- NOAEL
- Remarks:
- Reproductive toxicity
- Effect level:
- 3 225 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no effects on gestational length
- Dose descriptor:
- NOAEL
- Remarks:
- Reproductive toxicity
- Effect level:
- 300 mg/kg bw/day
- Based on:
- element
- Remarks:
- Al 3+
- Sex:
- female
- Basis for effect level:
- other: no effects on gestational length
- Key result
- Critical effects observed:
- no
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 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. - Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- Higher mortality and significant morbidity were observed in the male pups in the high dose group.
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. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 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 and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- 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. - Sexual maturation:
- effects observed, treatment-related
- Description (incidence and severity):
- 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. - Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- 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. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Necropsy Results
Urinary tract pathology (hydronephrosis, ureteral dilation, obstruction and/or presence of calculi) was an unexpected finding more prevalent in males and in the high dose group. The calculi (“chalky white concretions and deposits”) varied from sand-like material to stones up to 4 mm in diameter. Hyperkalemia was proposed by the pathologist as the cause of death of the animals with urinary obstruction. The chemical composition of the calculi was not determined.
The numbers of rats per cohort and treatment group that exhibited urinary tract pathology (hydronephrosis, ureteral dilation, obstruction and/or presence of calculi) are provided in the tables below (data extracted from Table 4 of the final report):
Females
Group/Cohort Day 23 Day 64 Day 120 Day 364
Control 0 1 0 0
Low Dose 0 0 0 0
Mid-Dose 0 1 0 0
High-Dose 0 3 2 3
Na-citrate 0 0 1 0
Males
Group/Cohort Day 23 Day 64 Day 120 Day 364
Control 0 0 0 0
Low Dose 0 0 0 1
Mid-Dose 0 3 1 0
High-Dose 0 11 7 5
Na-citrate 0 1 0 0
Urinary tract pathology was a treatment-related effect.
The only other treatment-related effect reported was watery, tan-coloured fluid in the digestive tract in some high dose animals, more frequently in the Day 64 group. - Histopathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 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. - Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- FOB (neonatal pups)
Females
No convulsions, salivation, and tremor were observed in females. No significant group effects were observed for activity, foot-splay, lacrimation, posture, unusual appearance or unusual behaviour.
Males
No convulsions, posture, salivation, tremor and unusual behaviour were observed 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. - Dose descriptor:
- NOAEL
- Remarks:
- Repeated Dose Toxicity-Neuromuscular effects
- Generation:
- F1
- Effect level:
- 322.5 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: LOAEL = 1075 mg AlCitrate/kg bw/day
- Dose descriptor:
- NOAEL
- Remarks:
- Repeated Dose Toxicity-Neuromuscular effects
- Generation:
- F1
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- element
- Remarks:
- Al 3+
- Sex:
- male/female
- Basis for effect level:
- other: LOAEL = 100 mg Al/kg bw/day
- Dose descriptor:
- LOAEL
- Remarks:
- Repeated Dose Toxicity-Neuromuscular effects
- Generation:
- F1
- Effect level:
- 1 075 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: hindlimb grip strength, forelimb footsplay
- Dose descriptor:
- LOAEL
- Remarks:
- Repeated Dose Toxicity-Neuromuscular effects
- Generation:
- F1
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- element
- Remarks:
- Al 3+
- Sex:
- male/female
- Basis for effect level:
- other: hindlimb grip strength, forelimb footsplay
- Key result
- Critical effects observed:
- no
- Reproductive effects observed:
- not specified
- Conclusions:
- The results from this study are informative for developmental and neurotoxic effects due to prenatal and chronic postnatal exposure of rats to high doses of aluminium (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.
Under the test conditions, the NOAEL for maternal toxicity is set at 300 mg Al/kg bw/day and the NOAEL for F1 generation is set at 30 mg Al/kg bw/day. - Executive summary:
This study was designed “to develop data on the potential functional and morphological hazards to the nervous system that may arise from pre-and post-natal exposure to aluminium citrate”. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions of aluminium citrate at 3 dosage levels (nominal - 30, 100 and 300 mg Al/kg bw/day. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.
Endpoints and observations in the dams included water consumption, body weight, a Functional Observational Battery (FOB), morbidity and mortality. Endpoints were assessed in both female and male pups that targeted behavioral ontogeny (motor activity, T-maze, auditory startle, the Functional Observational Battery (FOB) with domains targeting autonomic function, activity, neuromuscular function, sensimotor function, and physiological function), cognitive function (Morris swim maze), brain weight, clinical chemistry, haematology, tissue/blood levels of aluminium and neuropathology at the different dose levels and time points PND 23, 64, 120 and 364.
Statistical analyses were undertaken according to intention-to-treat, with appropriate consideration of multiple testing issues and, through the study design, also the unit of analysis. Censored analyses using survival analysis (Fixed Effects Partial Likelihood) were required for the grip strength measurements due to an equipment-defined maximum value. Females and males were analysed separately.
There were no significant Al-citrate treatment-related effects on mean body weights observed in the dams during the gestation and postnatal periods. The Na-citrate group, however, was significantly lighter than the control group on PND 15 (7.3%; p=0.0316). Eight dams in the high dose aluminium group were found to have diarrhoea compared with none in the other treatment groups. The low and mid-dose Al-citrate groups consumed more water than the control group but the high dose group did not, suggesting that the effect was not simply due to treatment. There were no significant treatment-related differences in gestational length. There were no consistent treatment-related effects observed for the FOB tests in the dams. Due to the differences in water consumption, the % of target dose differed between groups and with time through the study. In the high dose group of dams, the actual dose during the first week of gestation was 200 mg Al/kg bw/day, 67% of the target dose (300 mg Al/kg bw/day). In the last week before weaning (and sacrifice), the actual dose received by the dams was close to 175% of the target dose. Statistical analyses comparing the actual doses received by the low, mid- and high- Al-citrate treatment groups showed that the order of the dose groups was maintained, however.
The most notable treatment-related effect observed in the offspring was renal pathology – hydronephrosis, ureteral dilation, obstruction and presence of calculi - most prominently in the male pups. Higher mortality and significant morbidity were observed in the male pups in the high dose group; leading to euthanization of this group at ca. study day 89. Clinical observations that showed a relationship with treatment, either directly or secondary to renal failure, were poor coat, weight loss, and haematuria. Diarrhoea was also observed. These signs were found only in the high dose Al-citrate treatment group. Haematuria was also observed in some animals in the Na-citrate group in the Day 364 cohort. Dosing with Al-citrate was associated with a reduction in body weight. The results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. In the Day 120 cohort male pups, the mid-dose animals were significantly lighter than the controls. An effect of Na-citrate was observed in the female pups in the Day 364 cohort. Overall, dosing of animals with aluminium citrate led to higher fluid consumption than in the control animals. Dosing with Na-citrate was associated with a significant increase in fluid consumption relative to that of the controls in most cohorts, with the exception of the Day 64 cohort females (fluid consumption was significantly lower in the Na-citrate group) and the Day 364 males (no significant difference between the two groups). The animals’ fluid consumption varied with time and, in mature animals, was less than expected (120 mL/kg bw/day) with implications for the actual dosage of test item received. Despite the deviations from the target dose, the low-, mid- and high-dose groups showed the required trend of lowest to highest maintaining statistically significant group differences in dose levels. For most of the study period, the actual dose received was less than the target dose in all treatment groups.
In the female pups, the mean number of days to reach vaginal opening was 31.3 (±2.1, sd) in the control group and 39.7 (±5.6, sd) in the high dose Al-citrate group, a significant difference (p<0.0001). In males, the mean number of days to reach preputial separation was 39.6 (±2.1, sd) in the control group and 42.5 (±3.2, sd) in the high dose group, also a significant difference in the pair-wise comparisons (p<0.0001). Delayed development of both male and female pups was observed in the high dose Al-citrate and Na-citrate groups. The effect is considered treatment-related but whether the effect is secondary to decreases in body weight is not clear, however.
FOB observations showed no clear treatment-related effect among the neonatal Day 364 cohort pups that were assessed at PND 5 and 11 or in the juvenile pups assessed ca.PND 22. In the adult pups, the data provide little evidence for an Al effect on the autonomic function domain, the sensimotor function domain, or excitability. Significant wasting (physiological domain), was observed in the high dose females and appears related to treatment. Characteristics of defecation (number of boluses) also showed differences with treatment. In addition, there was limited evidence of effects on activity/well-being of the pups at the high dose as reflected in fur appearance, deposits and rearing. There was some evidence for dose-response relationships between neuromuscular measurements – hind-limb and fore-limb grip strength and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. Although the FOB endpoint most consistently associated with Al-citrate treatment, grip strength, measurements showed considerably variability and a consistent ordering of the Al-treatment group responses (dose-response) was not observed at all time points. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning Day 23 cohort) or the Morris Water Maze test (Day 120 cohort).
Haematology parameters showed no significant treatment-related effects in the Day 23 cohort. In the Day 64 cohort, however, both males and females showed low grade microcytic anaemia (significantly lower mean cell volume, mean cell haemoglobin, and haematocrit). The anaemia had resolved by the end of the study in the Day 364 cohort females. Clinical chemistry results showed serum chemistry changes associated with aluminium toxicity such as elevated alkaline phosphatase and serum calcium. The authors state the levels still remained within the normal range. Effects were most pronounced in the Day 64 cohort animals. By Day 364 in the females, alkaline phosphatase levels did not differ significantly between the treatment groups.
Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control groups. There were no significant sex differences. These results suggest transfer of Al from dams to pups in utero, although a contribution from breast milk PND 0 to 4 is also possible. Aluminium levels were assayed in several tissues in the pup cohorts. Levels of Al in whole blood were highest in the Day 23 cohort animals and declined with time, possibly due to the lower amounts of water (test solution) consumed once the pups matured. Although during the lactation period pups may have consumed some water/test solution, the results suggest that transfer of Al from dams to pups can occur through breast milk. Concentrations of Al in bone showed the strongest association with Al dose and some evidence of accumulation over time in all of the Al-treated groups. Of the central nervous system tissues, Al levels were highest in the brainstem. Although levels of Al were relatively low in the cortex (< 1µg/g), they were positively associated with Al levels in the liver and femur. In females, Al levels in the high dose group remained elevated relative to the other groups at all time points suggesting that accumulation might have occurred.
Pathological examinations showed clearly that urinary tract pathology was a treatment-related effect. The only other treatment-related effect reported on necropsy was watery, tan-coloured fluid in the digestive tract in some high dose animals, more frequently in the Day 64 group.None of the lesions seen on histopathological examination of brain tissues of the Day 364 group was treatment-related and, as these were also seen in the control group, were likely due to ageing.
This study has much strength. It was conducted according to GLP with a design based on OECD TG #426. The study used adequate numbers of animals and randomization to reduce bias, assessed endpoints in both female and male offspring, and studied a wide range of neurotoxicity endpoints. Haematology, clinical chemistry, pathology and general toxicity endpoints were also assessed. Three dose levels were used although the highest was close to the MTD. Although representative of actual human exposures, extending the period of exposure beyond weaning until day 364 leads to ambiguity in interpretation of the results as effects observed later in the study may have resulted from either later exposures or exposures during periods critical for development. There were a number of deviations from protocol that are clearly described in the study report. The report mentions that the observation protocol was not always consistently followed and that the clinical observations for the Group B and E animals started on April 3, 2008 instead of March 19, 2008. Some auditory startle data was lost due to an equipment malfunction (16 animals in Cohort 1 and 7 animals in Cohort 2) resulting in a “loss of statistical power for this part of the study”. Some necropsies were not completed “22 pre-weaning animals (eight from one litter) and 3 post-weaning animals that died or were euthanized prior to scheduled sacrifice did not have a necropsy completed”. The equipment used to measure grip strength had a maximum capacity of 700g leading to the possibility of underestimation of grip strength in larger animals, particularly in the males. Overall, these deviations were unlikely to have impacted the results of the study.
The results from this study are informative for developmental and neurotoxic effects due to combined prenatal and chronic postnatal exposure of rats to high doses of aluminium (30 mg Al/kg bw/day, 100 mg Al/kg bw/day and 300 mg Al/kg bw/day).As the offspring were dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. Urinary tract pathology was observed in rats in the high dose group, more frequently and more severe in the males. The study showed no evidence of an effect of Al-citrate on memory or learning but a more consistent effect was observed in endpoints in the neuromuscular domain.
The ambiguity as to the critical period of exposure and the time-varying water consumption complicate the derivation of a point-of-departure from this study. A LOAEL of 100 mg Al/kg bw/day for aluminium toxicity is assigned. The critical effect was a deficit in fore- and hind-limb grip strength in the mid-dose group, supported by evidence of dose response and less consistently observed effects in the mid-dose animals: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools than controls); tail pinch (mid-dose females displayed more exaggerated responses); foot-splay (mid-dose females had significantly narrower foot-splay than the controls); and the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls).
Under the test conditions, the NOAEL for maternal toxicity is set at 300 mg Al/kg bw/day and the NOAEL for F1 generation pups is set at 30 mg Al/kg bw/day for thefunctional hazards to the nervous system (deficit in fore- and hind-limb grip strength in the mid-dose group).
Referenceopen allclose all
F0 and F1 males and females (50, 500 and 5000 ppm)
No treatment-related parental deaths or clinical signs of intoxication at any treatment groups in either male or female F0 and F1 rats.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
WATER CONSUMPTION
F0 males (50, 500 and 5000 ppm)
- was significantly lower during the entire 14-week treatment period in all three dose groups
F0 females (50ppm)
- was significantly lower than in controls during premating period (weeks 1,9 and 10 of dosing), 1 week of gestation and week 1 of lactation);
(500ppm)
- was significantly lower than in controls during premating, gestation and lactation period;
(5000 ppm)
- was significantly lower than in controls during premating, gestation and lactation period.
F1 males (50ppm)
- no significant changes were found compared to the control animals.
(500 and 5000 ppm)
- significantly lower than in controls throughout the treatment period
F1 females (50 ppm)
- was significantly lower than in controls during week 4 and 8-10 weeks of premating period;
(500, 5000 ppm)
- was significantly lower than in controls during premating, gestation and lactation period;
FOOD CONSUMPTION
F0 males (5000 ppm)
- was decreased in the 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F0 females (500 ppm)
- significantly decreased during week 1 of premating period.
(5000 ppm)
- significantly decreased at 1 week of premating period, and 2-3 weeks of lactation period.
F1 females (5000 ppm)
- significantly decreased at 2-3 weeks of lactation period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F1 males (50, 500 and 5000 ppm)
- no significant changes were observed in the Al treated and control groups.
BODY WEIGHT
F0 males (5000 ppm)
- significantly decreased in the first 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F0 females (5000 ppm)
- significantly decreased in the first 1 week of premating period and at the end of lactation period (21 day).
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F1 males (5000 ppm)
- significantly decreased in the first 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F1 females (5000 ppm)
- significantly decreased in the first 1-2 weeks of premating period;
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test compound was administered with drinking water. The mean aluminium ammonium sulfate and elemental Al intakes during the whole dosing period in F0 males, F0 females, F1 males and F1 females provided in Table 1 (any other information on materials and methods including tables).
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0 and F1 females (50, 500 and 5000 ppm)
estrous cycle
- during a premating period, a few AAS-treated F0 and F1 rats had persistent diestrus, however:
- no significant changes in the incidence of F0 and F1 females with normal estrous cycle (4-5 days) compared to the control animals were observed during the premating period;
- no significant differences in the estrous cycle between AAS treated and control groups were noted.
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 and F1 males (50, 500 and 5000 ppm)
There were no significant differences in the number of testis sperm and cauda epidymal sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm between control and AAS-treated groups in either F0 or F1 adults (however, no details were provided on the results of performed examination).
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance
F0 and F1 parental animals
The authors reported that some animals failed to copulate, impregnate or deliver live pups, however:
- no significant differences were observed between the control and AAS-treated groups in either F0 and F1 generation (24 animals per group) for copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations, number of litters or pups delivered. Overall, there were no treatment-related effects on reproduction parameters.
ORGAN WEIGHTS (PARENTAL ANIMALS)
Adults (F0 and F1)
- relative kidney weight was significantly increased in F0 females (500 and 5000ppm) and F1 males and females (5000ppm);
- absolute weight of the pituitary glands was significantly decreased in F0 females and F1 males and females (5000ppm);
- absolute thymus decreased significantly in F1 females(5000ppm);
- relative weight of seminal vesicle was significantly decreased in F1 males (50 ppm);
- absolute brain weight was significantly decreased in F1 females (500 ppm).
GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 males and females, F1 males and females
No AAS treatment- related gross lesions were observed in either generation.
HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 males and females, F1 males and females
No treatment related histopathological changes of the reproductive organs were observed.
F1 and F2 generation (50, 500 and 5000 ppm)
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation
CLINICAL SIGNS (OFFSPRING)
F1 generation
Clinical (external gross) examination during the preweaning period revealed microphthalmia, a rudimentary tail, trauma and scab on right hindlimb and crushing of incisor/malocclusion in a few F1 pups in control and AAS-treated groups (no data provided); however, there no significant differences in incidence between the control and AAS-treated animals were observed (data not shown);
F2 generation
No gross abnormalities in F2 pups were found in any groups.
BODY WEIGHT (OFFSPRING)
F1 generation (5000ppm group, F1 males and F1 females)
- body weight of male and female pups was significantly lower on PND 21 and on PND 14 and 21, respectively, compared to the control pups.
F2 generation (5000ppm, F2 males and females)
- body weights were lower than controls around time of weaning but no statistically significant differences were found.
SEXUAL MATURATION (OFFSPRING)
F1 females (5000 ppm)
vaginal opening
- was significantly delayed (32.3±1.8 days of age, compared to 30.2±2.1days of age in control). Body weight at the time of vaginal opening was slightly higher than the control (122.0±15.7 g. versus 115.8±12.6 g.) but no statistically significant difference was found.
F1 males (50, 500 and 5000 ppm)
the age at preputial separation
- no significant differences between control and AAS-treated groups were found and no changes were found in body weight at the time of completion of separation.
ORGAN WEIGHTS (OFFSPRING)
F1 males (Table 2) (5000 ppm)
Body weight was significantly decreased (85.97% compared to the control group); relative brain weight was significantly increased; absolute kidney weight was significantly decreased but the relative kidney weight was significantly increased ; absolute and relative thymus weight was significantly decreased;
absolute and relative liver weight was significantly decreased; absolute and relative spleen weight was significantly decreased; absolute weight of the adrenal glands was decreased significantly; absolute weight of testis and epididymis was significantly decreased.
F1 females (Table 3) (5000 ppm)
Body weight was significantly decreased (87.59% compared to the control group); relative brain weight was significantly increased; relative kidney weight was significantly increased; absolute thymus weight was significantly decreased; absolute liver weight was significantly decreased; absolute and relative spleen weight was significantly decreased; absolute weight of the adrenal glands was decreased significantly; absolute weight of uterus was significantly decreased.
F1 females (Table 3) (500 ppm)
weight of the adrenal glands was decreased significantly.
F2 males (Table 2) (5000 ppm)
Body weight was significantly decreased (92.21% compared to the control group); relative brain weight was significantly increased; absolute and relative thymus weight was significantly decreased; absolute liver weight was significantly decreased; relative kidney weight was significantly increased; absolute and relative spleen weight was significantly decreased.
F2 females (Table 3) (5000ppm)
Body weight was significantly decreased (90.50% compared to the control group); relative brain weight was significantly increased; absolute thymus weight was significantly decreased; absolute and relative liver weight was significantly decreased; relative kidney weight was significantly increased; absolute and relative spleen weight was significantly decreased; relative weight of the adrenal glands was increased significantly; absolute ovary weight was significantly decreased; absolute uterus weight was significantly decreased.
F1 females (5000ppm)
Number of primordial follicles in the ovary was no different between Al treated and control females (data not shown).
GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations
F1 males and females, F2 males and females
Gross observations did not reveal any compound-related lesions either in F1 and F2 weanlings or in pups found dead during the lactation period (no data provided that any pups were found dead during the lactation period).
HISTOPATHOLOGY (OFFSPRING)
F1 males and females, F2 males and females
No dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings and of the thymus in males in both generations.
OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT LANDMARKS
PHYSICAL DEVELOPMENT
F1 males/females and F2 males/females (50, 500 and 5000 ppm)
- Completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening , the AGD and AGD per cube root of the body weight ratio were not significantly different between the control and AAS-treated groups (data not shown).
NEUROMOTOR DEVELOPMENT
F1 males/females and F2 males/females (50, 500 and 5000 ppm)
- No significant changes were observed in the achieved day of the surface righting reflex (PND5), negative geotaxis reflex (PND8) and midair righting reflex (PND 18).
- No significant changes were observed in the response time of surface righting and negative geotaxis reflex (data not shown).
BEHAVIOR PERFORMANCE
SPONTANEOUS LOCOMOTOR ACTIVITY
F1 males (50, 500 and 5000 ppm)
- was not significantly different between control and AAS treated males at 10-min intervals and for 60 min;
F1 females (500 ppm)
Spontaneous locomotor activity was significantly decreased during the 40-50 and 50-60 minutes after start of test but no significant differences were observed in total activity for 60 min; no changes in spontaneous locomotor activity for 10 min intervals or for a total of 60 min between the control and the other AAS-treated groups in females
T MAZE TEST
Learning and memory performance in T-maze test (pre-test swimming trials in the straight channel)
F1 males and females (50, 500 and 5000 ppm)
- no significant changes were observed in the elapsed time to traverse the straight channel.
- no significant changes were observed in the elapsed time and number of errors on days 2–4.
Table1: Absolute and relative organ weight of F1 and F2 male weanlings (% to the control)
| AAS (ppm) | 0 | 50 | 500 | 5000 | ||||
| Organ weight | F1 males | F2 males | F1 males | F2 males | F1 males | F2 males | F1 males | F2 males |
| number of animals | 24 | 22 | 20 | 18 | 23 | 22 | 24 | 23 |
| body weigth (g) | 100% | 100% | 85.97** | 82.21* | ||||
brain |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 113.59** | 107.36* |
thymus |
||||||||
| absolute weigth (g) | 100% | 100% | NS | NS | NS | NS | 76.79** | 78.93** |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 89.21* | 86.09** |
liver |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 81.48** | 89.56* |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 94.76* | NS |
kidneya |
||||||||
| absolute weigth (g) | 100% | 100% | NS | NS | NS | NS | 90.74* | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 105.22* | 107.96** |
spleen |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 69.36** | 74.86** |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 80.76** | 80.92** |
adrenala |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 90.91* | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
testisa |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 90.02* | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
epididymisa |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 84.01** | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
NS- no statistically significant differences were observed in the effect between Al treated and control animals
**- significantly different from the control, p<0.01; *- significantly different from the control, p<0.05;
a- value represent the total weights of the organs on both sides
Table2: Absolute and relative organ weights of F1 and F2 female weanlings (% to the control group)
| AAS (ppm) | 0 | 50 | 500 | 5000 | ||||
| organ weight | F1 females | F2 females | F1 females | F2 females | F1 females | F2 females | F1 females | F2 females |
| number or animals | 24 | 22 | 21 | 18 | 23 | 22 | 24 | 23 |
| body weight (g) | 100% | 100% | 87.59** | 90.50** | ||||
brain |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 110 .88** | 108.21** |
thymus |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 82.72** | 81.74** |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
liver |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 86.54** | 84.73** |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | 93.49** |
kidneya |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 107.96** | 107.08 |
spleen |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 75.14** | 77.75** |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 85.57** | 86.06** |
adrenala |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | 89.01** | NS | 87.45** | NS |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 109.45* | |
ovarya |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | NS | 87.83** |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
uterus |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 74.89** | 75.35* |
| rel. weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
NS- no statistically significant differences were observed in the effect between Al treated and control animals
**- significantly different from the control, p<0.01; *- significantly different from the control, p<0.05;
a- value represents the total weights of the organs on both sides
No significant difference was seen between control and AS treated groups in the incidence of clinical signs of toxicity in either mal or female F0 and F1 rats.
120ppm: One F1 male died at 9 weeks of dosing. Soilin gof periocular and perinasal fur and decreased locomotor activity were observed before death. Necropsy revealed accumulation of ascitic and pleural fluid and dark purple discoloration of liver and kidneys
600ppm: One F0 female died at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this female from the beginning of 5 weeks of dosing
3000ppm: One F1 male died at 12 weeks of dosing. No clinical signs of toxicity were observed.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
drinking water consumption: F0 males and females - significantly lower than control throughout the study (120-3000 ppm); F1 males - 120 ppm: significantly decreased during 3-6, 8, 10 weeks of dosing; 600, 3000 ppm: significantly decreased through dosing period; F1 females - 120 ppm: significantly decreased during 9-10 weeks of dosing; 600 ppm: significantly decreased during 10 week of dosing and 3 week of lactation; 3000 ppm: significantly decreased throughout the dosing period compared to controls
Food consumption: F0 males - 600ppm: significantly reduced during the first week of dosing; 3000ppm: significantly decreased during weeks 1,8 and 13-14; F0 females - 600ppm: significantly decreased during week 3 of lactation; 3000ppm: significantly decreased at 1 week of dosing and during week 3 of lactation; F1 males and females - 600 and 3000 ppm: significantly decreased in the 10 week of dosing (F1 males); significantly decreased in the 3 week of lactation (F1 females);
body weight: F0 males and females - 3000 ppm: significantly decreased in the first 2 or 3 weeks of dosing; 120 (F1 males), 600 (F1 males and F1 females) and 3000 ppm (F1 males and F1 females) - no significant differences in body weight compared to control; 120 ppm, F1 females - significantly increased body weights during 6-8 weeks of dosing
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
se table1 under "any other information on material and methody including tables"
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0 and F1 females - 120, 600 and 3000 ppm:
estrous cycle: no significant deviations in the estrous cycle of F0 and F1 females were observed during the premating period. However, a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation.
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 males - 3000 ppm: absolute number of cauda epididymal sperm - reduced significantly (253.8 ±61.3 × 106/cauda versus 286.3 ±40.3 ×106/cauda in the control); however when expressed as the number per gram of tissues, there was no significant change.
F1 males - 3000 ppm: absolute number of cauda epididymal sperm - no change was found compared to the control animals. Number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm - no significant differences between control and AS-treated groups in either F0 or F1 adults (Note: no details were provided on the results of these examinations).
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0 and F1 parental generation - copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations, number of litters or pups delivered (see table2)
- no significant differences were observed between the control and AS-treated groups in either F0 and F1 generation.
Copulation was not observed:
- in the F0 males: control (n=2), 120 ppm (n=2), 3000 ppm (n=2) and in the F0 females: in the control (n=1);
- in the F1 males: in the control group (n=1), 120 ppm (n=2) 600 ppm (n=1) and 3000 ppm (n=3) and in the F1 females: in the 120 ppm group (n=1), 3000 ppm (n=1).
After successful copulation, no pregnancy was observed:
- in F0 females: in the control (n=1), 120 ppm (n=2), 3000 ppm (n=1);
- in F1 females: in the control (n=2), 120 ppm (n=4), 600 ppm (n=2) and 3000 ppm (n=2).
No live pups delivered were found for pregnant rats from :
- F0 female in the 120 (n=1), 600(n=1) and 3000 ppm (n=1);
- F1 female in the 120 ppm group (n=1).
Comments: Overall, there were no treatment-related effects on reproduction parameters.
ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 males - 3000 ppm
absolute and relative liver weights
- were significantly decreased;
absolute spleen weight
- was significantly decreased;
- no significant change in relative weight.
F1 males - 3000 ppm
absolute weight of the adrenals
- was significantly decreased;
- no significant change in relative weight.
F1 males - 600 ppm
absolute weight of the testes
- was significantly decreased;
- no significant change in relative weight.
F0 and F1 females - 120, 600 and 3000 ppm
- no changes in absolute or relative weights of organs compared to the control (data not shown).
F0 and F1 females - 3000 ppm
number of primordial follicles in the ovary
- no difference between AS-treated and controls (data not shown).
GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 and F1 generations - No dose-related gross lesions were found in F0 or F1 adults.
HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 and F1 males and females - 3000 ppm:
Histopathological examination of the reproductive organs revealed no compound-related alterations.
OTHER FINDINGS (PARENTAL ANIMALS)
F1 and F2 generation - 120, 600 and 3000 ppm:
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation.
CLINICAL SIGNS (OFFSPRING)
F1 generation - During the in-life check of delivered pups, one control F1 pup experienced trauma in the perianal region and tail and one F1 pup had hemimelia and oligodactyly in the 120ppm group, but there was no significant difference between the control and AS-treated groups.
F2 generation - No malformed F2 pups were found in any group.
BODY WEIGHT (OFFSPRING)
F1 generation - 3000 ppm group, F1 males and F1 females
- body weights of male and female pups were significantly lower on PND 21 compared to the control.
F2 generation - 3000 ppm, F2 females
- body weights were significantly lower than controls on PND 21.
3000 ppm, F2 males
- there were no significant differences in body weights between the control and AS-treated groups during the preweaning period.
SEXUAL MATURATION (OFFSPRING)
F1 males and females - 3000 ppm
- vaginal opening was significantly delayed (31.4±1.7 compared to 29.5±2.1 days in control). At 3000 ppm body weight at the time of vaginal opening was slightly higher than the control (119.0 ± 13.3 versus 109.6 ± 11.6 g) although not statistically significant.
- 120, 600 and 3000 ppm
- no significant differences between control and AS-treated groups were noted regarding age at preputial separation and no changes were found in body weights at the time of preputial completion.
ORGAN WEIGHTS (OFFSPRING) (see table3 and 4)
F1 generation - 3000 ppm, males and females
body weight - significantly lower at scheduled sacrifice compared to the control;
absolute and relative liver weights - significantly lower than the control;
absolute spleen weight - significantly decreased in both males and females and, a significant decrease in the relative weight was observed in males;
the absolute weight of the thymus - decreased in both sexes;
absolute weight of the kidney, testes and epididymides (males) - decreased compared to the control;
absolute weight of the uterus - decreased at 600 ppm compared to control;
relative brain weight - significantly increased in both sexes.
F2 generation - 3000 ppm, males
mean body weight at sacrifice - significantly lowered in both sexes;
absolute and relative weights of the thymus and spleen - significantly decreased in males;
absolute weight of the liver and epididymides - significantly decreased;
relative brain weight - significantly increased.
120 ppm, males
relative thymus weight - significantly decreased but no dose-response relationship.
3000 ppm, females
absolute and relative weights of the liver, the absolute weights of the spleen, ovary and uterus - significantly decreased;
relative brain weight - significantly increased.
600 ppm, females
the absolute brain weight - significantly decreased.
GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations:
F1 males and females, F2 males and females
- no treatment-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period (data not shown).
HISTOPATHOLOGY (OFFSPRING)
F1 males and females, F2 males and females
- no dose-related histopathological changes in the liver or spleen of male and female F1 and F2 weanlings. (fpr details see table 3+4)
OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT:
F1 males and females; F2 males - 120, 600 and 3000 ppm
- the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening were not significantly different between the control and AS-treated groups.
F1 males and females, F2 males and females - 120, 600 and 3000 ppm
- the AGD and AGD per cube root of the body weight ratio were not significantly different between control and AS-treated groups in male and female F1 and F2 pups (data not shown).
F2 females - 120, 600 and 3000 ppm
-completion rates of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks were not significantly different between AS-treated groups and controls.
600ppm - completion rates of pinna unfolding on PND 2 was significantly lower (17.0±35.4%, compared with 45.8±46.9 in controls), but no dose-response relation was observed.
NEUROMOTOR DEVELOPMENT
F1 males and females - 120, 600 and 3000 ppm
- no significant changes were observed in the development of reflexes (surface righting reflex on PND5, negative geotaxis reflex on PND8 and midair righting reflex on PND 18);
- no significant changes were observed in the response times of surface righting and negative geotaxis reflexes.
F2 males and females - 120, 600 and 3000 ppm
- surface righting reflex on PND 5 and negative geotaxis reflex on PND 8 were achieved in all male and female F2 pups in all groups;
- no significant changes were found in the response time (data not shown).
F2 females - 600 ppm
- the mid-air righting reflex on PND 18 was not achieved by 1 female in one of three trials; however, there was no significant difference in the mean success rate between the control and 600 ppm group (100±0.0% versus 98.4±7.3%).
BEHAVIOR PERFORMANCE
F1 males and females - 120, 600 and 3000 ppm
Spontaneous locomotor activity was not significantly different between control and AS- treated groups at 10-min intervals and for 60 min.
Learning and memory performance in T-maze test
Pre-test swimming trials in the straight channel
- no differences between male and female rats in each group compared to the controls;
- no significant changes in the elapsed time to traverse the straight channel;
- in males, no significant changes in the elapsed time and number of errors on days 2–4;
- in females, the elapsed time and the number of errors was significantly lowered at 600 ppm on day 2, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 between control and AS-treated groups (data not shown).
Table2: Reproductive performance of F0 and F1 parental animals
| AS (ppm) | 0 | 120 | 600 | 3000 | |
| F0 generation | |||||
| No. of rats (male/female) | 24/24 | 24/24 | 24/24 | 24/24 | |
| Copulation index (%) | males | 91.7 | 91.7 | 100 | 91.7 |
| females | 95.8 | 100 | 100 | 100 | |
| Precoital interval (days) | 3.2 ± 1.1 | 3.2 ± 1.8 | 2.9 ± 1.3 | 2.8 ± 1.6 | |
| Fertility index (%) | males | 95.5 | 90.9 | 100 | 95.5 |
| females | 95.7 | 91.7 | 100 | 95.8 | |
| Gestation index (%) | 100 | 95.5 | 95.7 | 95.7 | |
| Gestation length (days) | 22.4 ± 0.5 | 22.5 ± 0.6 | 22.1 ± 0.4 | 22.3 ± 0.5 | |
| Delivery index (%) | 94.3 ± 5.6 | 88.6 ± 21.0 | 90.7 ± 20.8 | 92.0 ± 20.5 | |
| F1 generation | |||||
| No. of rats (male/female) | 24/24 | 23/24 | 24/24 | 24/24 | |
| Copulation index (%) | males | 95.8 | 91.3 | 95.8 | 87.5 |
| females | 100 | 95.8 | 100 | 95.8 | |
| Precoital interval (days) | 3.3 ± 3.2 | 3.0 ± 2.0 | 2.7 ± 1.5 | 2.3 ± 1.1 | |
| Fertility index (%) | males | 91.3 | 81.0 | 91.3 | 95.2 |
| females | 91.7 | 82.6 | 91.7 | 91.3 | |
| Gestation index (%) | 100 | 94.7 | 100 | 100 | |
| Gestation length (days) | 22.4 ± 0.5 | 22.3 ± 0.5 | 22.2 ± 0.4 | 22.2 ± 0.4 | |
| Delivery index (%) | 94.0 ± 9.9 | 87.5 ± 22.6 | 91.4 ± 10.7 | 94.6 ± 6.8 |
Table3: Absolute and relative organ weight of F1 and F2 male weanlings (% of control)
| As (ppm) | 0 | 120 | 600 | 3000 | ||||
| Organ weight | F1 males | F2 males | F1 males | F2 males | F1 males | F2 males | F1 males | F2 males |
| number of animals | 22 | 21 | 20 | 18 | 22 | 22 | 22 | 21 |
| body weight (g) | 100% | 100% | NS | NS | NS | 87.44** | 90.31** | |
| brain | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 113.22** | 112.11** |
| thymus | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | 81.33** | 79.84** | |
| relative weight (g/100g bw) | 100% | 100% | NS | 89.29* | NS | NS | NS | 87.92** |
Livera |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 80.60** | 87.78** |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 91.61** | NS |
| Kidneya | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 89.62** | NS |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| spleen | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 76.40** | 80 .43 |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 86.93** | 88.36** |
| testisa | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 90.44* | NS |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| Epididymisa | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 88.02** | 93.62* |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
NS- not statistically significant compared to untreated control
**- significantly different from control, p<0.05
*- significantly different from control, p< 0.01
Table4: Absolute and relative organ weight of F1 and F2 female weanlings (% of control)
| As (ppm) | 0 | 120 | 600 | 3000 | ||||
| Organ weight | F1 females | F2 females | F1 females | F2 females | F1 females | F2 females | F1 females | F2 females |
| number of animals | 22 | 22 | 20 | 18 | 22 | 21 | 21 | 21 |
| body weight (g) | 100% | 100% | NS | NS | 89.91** | 91.34** | ||
| brain | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | 102.5* | NS | NS |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | 110.20** | 110.05** | |
| thymus | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 81.72** | NS |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
Livera |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 84.80** | 86.24** |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | 94.26* | 94.56** |
| Kidneya | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| spleen | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | 86.65** | 84.11 |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
| ovarya | ||||||||
| absolute weight (g) | 100% | 100% | NS | NS | NS | NS | NS | 84.52** |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
uterusa |
||||||||
| absolute weight (g) | 100% | 100% | NS | NS | 83.85* | NS | 78.47** | 81.49* |
| relative weight (g/100g bw) | 100% | 100% | NS | NS | NS | NS | NS | NS |
NS- not statistically significant compared to untreated control
**- significantly different from control, p<0.05
*- significantly different from control, p< 0.01
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)
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
1 female of the control group sacrified in extremis at day 22 of pregnancy due to parturition difficulties
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.
The target dose for the low dose group was 30 mg Al/kg bw/day, for the mid-dose 100 mg Al/kg bw/day and for the high dose 300 mg Al/kg bw/day. The table below provides the arithmetic mean actual dose as a % of the target dose for 5 selected post-weaning weeks in the Day 364 cohorts.
Males
Group Week1 Week7 Week14 Week28 Week49
Low Dose 134% 57% 37% 20% 17%
Mid-Dose 174% 84% 51% 28% 23%
High-Dose 165% 117% - - -
Females
Group Week1 Week7 Week14 Week28 Week49
Low Dose 145% 60% 57% 34% 33%
Mid-Dose 199% 74% 64% 38% 41%
High-Dose 205% 118% 93% 58% 42%
Despite the deviations from the target dose, the low-, mid- and high-dose groups showed the required trend of lowest to highest maintaining statistically significant group differences in dosage. For the majority of the study period, the actual dose received was less than the target dose in all treatment groups.
Morris Water Maze
Training Trial Latencies
There were no significant effects of treatment group in males or females for the Day 64 cohorts, the Day 120 cohorts or the Day 364 cohorts.
Platform-Removed Probe Test Search Strategies
No significant treatment group effects in either sex or any of the cohorts.
Platform Visible Latencies
No significant treatment group effects in either sex or any of the cohorts.
Platform Visible Type of Search
No significant treatment group effects in either sex or any of the cohorts.
Overall, there was no evidence for effects of aluminium on animal performance in the Morris Water Maze Test. Haematology
Day 23 cohort, females: The low dose group had significantly lower mean cell volume (MCV) than the control group (p=0.0189). The platelet count (PLT) was significantly lower in the low dose group than in the high dose group (p=0.0418). Nucleated red blood cells (NUC-RBC) in the low dose group differed significantly from this parameter in the control, mid-dose and high dose groups (p=0.0363, p=0.0101, and p=0.0062, respectively).
Day 23 cohort, males: The high dose group had marginally higher MCV than the control group (p=0.050).
Day 64 cohort, females:
Day 64 cohort, males:
Parameter |
Pairwise Differences |
Absolute Agranulocytes |
Ns |
Absolute Granulocytes |
The high dose group was significantly greater than the controls and low dose group (p=0.0240 and p=0.0354, respectively) |
Agranulocytes |
Significant group effect but no pair-wise comparisons with p-values<0.05. |
Granulocytes |
Significant group effect but no pair-wise comparisons with p-values<0.05. |
HCT (haematocrit) |
The high dose group was significantly lower than the controls and the low dose group (p=0.0113 and p=0.0238, respectively). The Na-citrate group was significantly lower than the control group (p=0.0365). |
HGB (haemoglobin) |
The high dose group was significantly lower than the control and the low dose group (p=0.0181 and p=0.0202, respectively). |
MCH (mean cell haemoglobin) |
The high dose group was significantly lower than all the other groups (controls, p<0.0001; low dose group, p=0.0009; mid-dose group, p=0.0005; Na-citrate group, p=0.0010). |
MCHC (mean cell haemoglobin concentration) |
Ns |
MCV (mean cell volume) |
The high dose group was significantly lower than all the other groups (controls, p<0.0001; low dose group, p=0.0007; mid-dose group, p=0.0005; Na-citrate group, p=0.0012). |
PLT (platelet count) |
Ns |
NUC_RBC (nucleated red blood cells) |
Zero |
RBC (red blood cell count) |
The high dose group was significantly greater than the mid-dose group (p=0.0341) and the Na-citrate group (p=0.0034). |
WBC (white blood cell count) |
Ns |
Day 120 cohort, females: Absolute levels of granulocytes and agranulocytes were significantly elevated in the high dose group relative to the control, low- and mid-dose groups. MCH was significantly lower in the high dose group than in the control, mid-dose, and Na-citrate groups. Similar to the Day 64 cohort results, the MCV was significantly lower in the high dose group than in all other treatment groups also. The white blood cell count was significantly higher in the high dose group compared to that in the control, the low-dose and the mid-dose groups.
Day 120 cohort, males: High dose males had been euthanized at this point. The only significant inter-group difference was for MCV. Levels were significantly lower in the Na-citrate group than in the controls (p=0.0260).
Day 364 cohort, females: No significant effects of group.
Day 364 cohort, males: No significant effects of group.
Overall, effects in the Day 23 cohort were not considered clinically significant. In the Day 64 cohort, however, both males and females in the high dose group showed low grade microcytic anaemia. The anaemia had resolved in the females by cohort Day 364.
Coagulation parameters:
No significant treatment group effects were found for the coagulation parameters.
Clinical Chemistry
Clinical Chemistry – Serum Parameter Values in the control groups (10 animals/group)
Female Controls (mean (standard deviation))
Parameter |
Units |
Day 23 |
Day 64 |
Day 120 |
Day 364 |
ALB (albumin) |
g/L |
34.5 (1.51) |
45.00 (1.89) |
50.27 (2.33) |
48.25 (3.62) |
ALP (alkaline phospha-tase) |
U/L |
330.30 (36.32) |
119.20 (21.40) |
52.91 (19.03) |
36.25 (18.12) |
ALT (alanine aminotrans-ferase) |
U/L |
28.80 (4.32) |
23.70 (5.46) |
20.45 (4.55) |
25.00 (3.55) |
AST (aspartate aminotrans-ferase) |
U/L |
173.10 (48.21) |
81.00 (17.40) |
74.55 (9.68) |
108.88 (44.96) |
A_G (albumin/ globulin ratio) |
|
2.55 (0.33) |
2.68 (0.31) |
2.52 (0.16) |
1.95 (0.30) |
CA (calcium) |
mM |
2.86 (0.05) |
2.76 (0.08) |
2.71 (0.08) |
2.67 (0.10) |
CHOL (cholesterol) |
mM |
2.60 (0.39) |
2.09 (0.49) |
1.85 (0.38) |
3.68 (0.86) |
CK (creatinine kinase) |
U/L |
972.20 (479.79) |
414.30 (109.88) |
308.55 (132.96) |
438.25 (336.60) |
CL (chloride) |
mM |
101.40 (2.17) |
99.60 (2.80) |
102.64 (1.03) |
100.00 (1.41) |
CRE (creatinine) |
µM |
12.70 (5.40) |
29.20 (3.97) |
42.27 (6.68) |
41.13 (4.97) |
GLOB (globulin) |
g/L |
13.70 (1.64) |
17.00 (2.00) |
20.00 (1.41) |
25.00 (2.33) |
GLU (glucose) |
mM |
10.18 (1.27) |
12.80 (1.68) |
11.15 (1.11) |
9.25 (2.09) |
K (potassium) |
mM |
5.11 (0.24) |
4.22 (0.38) |
4.55 (0.42) |
4.30 (0.44) |
Na (sodium) |
mM |
137.40 (1.71) |
141.00 (2.45) |
141.55 (2.21) |
144.88 (2.70) |
Phos (phosphorus) |
mM |
2.66 (0.22) |
2.23 (0.39) |
1.92 (0.25) |
1.73 (0.35) |
SDH (Sorbitol dehydrog-enase) |
U/L |
52.10 (9.50) |
35.30 (7.20) |
36.09 (17.54) |
66.25 (21.53) |
TBIL (total bilirubin) |
µM |
1.50 (0.53) |
2.00 (0.47) |
2.73 (0.47) |
2.75 (0.46 ) |
TG (triglycerides) |
mM |
1.62 (0.53) |
1.85 (0.82) |
3.91 (3.42) |
6.16 (6.52) |
TP (total protein) |
g/L |
48.20 (2.04) |
62.00 (3.23) |
70.27 (3.23) |
73.25 (3.11) |
Urea |
mM |
5.99 (1.20) |
6.14 (1.26) |
4.95 (0.58) |
5.38 (1.08) |
Male Controls (mean (standard deviation))
Parameter |
Units |
Day 23 |
Day 64 |
Day 120 |
Day 364 |
ALB (albumin) |
g/L |
34.40 (1.65) |
37.60 (1.90) |
38.67 (3.24) |
36.00 (4.90) |
ALP (alkaline phospha-tase) |
U/L |
332.50 (51.70) |
203.30 (33.45) |
87.78 (15.78) |
70.00 (16.84) |
ALT (alanine aminotrans-ferase) |
U/L |
26.80 (4.54) |
29.50 (7.85) |
29.56 (12.64) |
57.00 (39.55) |
AST (aspartate aminotrans-ferase) |
U/L |
151.70 (12.98) |
105.00 (21.85) |
83.78 (16.32) |
134.75 (53.51) |
A_G (albumin/ globulin ratio) |
|
2.49 (0.21) |
1.97 (0.21) |
1.58 (0.14) |
1.23 (0.21) |
CA (calcium) |
mM |
2.85 (0.08) |
2.72 (0.10) |
2.65 (0.04) |
2.66 (0.14) |
CHOL (cholesterol) |
mM |
2.47 (0.31) |
1.92 (0.41) |
2.03 (0.33) |
3.70 (1.32) |
CK (creatinine kinase) |
U/L |
806.10 (190.93) |
633.40 (149.19) |
387.33 (152.60) |
557.50 (174.88) |
CL (chloride) |
mM |
99.70 (2.00) |
99.10 (1.66) |
102.33 (1.12) |
101.25 (1.39) |
CRE (creatinine) |
µM |
10.60 (3.81) |
19.90 (4.09) |
30.11 (5.46) |
40.63 (9.10) |
GLOB (globulin) |
g/L |
13.90 (1.10) |
19.30 (2.11) |
24.56 (0.73) |
29.50 (2.83) |
GLU (glucose) |
mM |
8.88 (1.22) |
12.49 (2.24) |
12.74 (1.82) |
9.60 (1.22) |
K (potassium) |
mM |
5.00 (0.35) |
4.57 (0.30) |
4.49 (0.29) |
4.94 (0.49) |
Na (sodium) |
mM |
136.80 (1.75) |
141.70 (1.42) |
142.33 (1.00) |
146.00 (3.89) |
Phos (phosphorus) |
mM |
2.51 (0.19) |
2.60 (0.26) |
2.05 (0.13) |
2.03 (0.47) |
SDH (Sorbitol dehydrog-enase) |
U/L |
51.90 (8.70) |
52.70 (17.95) |
33.44 (15.80) |
72.50 (39.58) |
TBIL (total bilirubin) |
µM |
1.30 (0.48) |
1.70 (0.48) |
2.56 (0.53) |
3.13 (1.46) |
TG (triglycerides) |
mM |
2.10 (1.23) |
2.20 (0.51) |
3.13 (1.07) |
2.96 (1.41) |
TP (total protein) |
g/L |
48.30 (2.11) |
56.90 (3.14) |
63.22 (3.35) |
65.50 (5.48) |
Urea |
mM |
5.23 (1.23) |
7.07 (1.26) |
4.88 (0.70) |
5.74 (1.34) |
Statistically significant differences from the pair-wise comparisons are provided in the table below. Pair-wise comparisons were only conducted where a significant effect of group was found in the ANOVA. Results from comparisons between the control and the different aluminium citrate groups are in bold font.
FEMALES
Parameter |
Day 23 |
Day 64 |
Day 120 |
Day 364 |
ALB (albumin) |
Log transform-ation required.
|
High < control (p=0.0002), low (p=0.0014) and mid dose (p=0.0005). |
High dose < low (p=0.0087) and mid dose (p=0.0028) groups. |
|
ALP (alkaline phospha-tase) |
|
High> control, low-dose and mid-dose (p<0.0001)
High dose>Na-citrate (p<0.0001) |
High dose > control (p=0.0013), low dose (p=0.0071), and mid-dose (p=0.0300) |
|
ALT (alanine aminotrans-ferase) |
|
|
|
|
AST (aspartate aminotrans-ferase) |
Log transformed.
|
|
|
|
A_G (albumin/ globulin ratio) |
|
|
|
|
CA (calcium) |
High > control (p=0.0117).
Na-citrate group < mid dose (p=0.0038) and high dose groups (p=0.0001). |
High> control, low-dose and mid-dose (p<0.0001)
High dose>Na-citrate (p<0.0001) |
High > control (p=0.0201).
High > Na-citrate (p=0.0045) |
|
CHOL (cholesterol) |
|
|
|
|
CK (creatinine kinase) |
|
|
|
|
CL (chloride) |
|
|
Na-citrate < control (p=0.0051) |
Na-citrate < control (p=0.0038) and low dose (p=0.0256) |
CRE (creatinine) |
|
All adjusted p values >0.05. |
All adjusted p-values <0.05) |
|
GLOB (globulin) |
|
High < control (p=0.0026), low-dose (p=0.0189) and mid-dose (p=0.0004).
High dose<Na-citrate (p=0.0484) |
High < mid dose (p=0.0339) |
|
GLU (glucose) |
Control > high dose group (p=0.0214) & low dose group (p=0.0447).
Na-citrate < control (p=0.0007) |
All adjusted p-values > 0.05 |
|
|
K (potassium) |
Control>low dose group (p=0.0463).
Na-citrate <control (p=0.0018) |
|
|
All adjusted p-values >0.05. |
Na (sodium) |
Na-citrate group > control (p<0.0001), low dose (p<0.0001), mid-dose (p<0.0001) and high dose (p=0.0069). |
Mid > high dose (p=0.0103)
Mid-dose > Na-citrate (p=0.0168). |
|
|
Phos (phosphorus) |
Control > high dose group (p=0.0009) |
|
|
|
SDH (Sorbitol dehydrog-enase) |
|
|
|
|
TBIL (total bilirubin) |
Categorical. |
|
|
|
TG (triglycerides) |
|
High dose < control (p=0.0047) and low dose (p=0.0145). |
|
|
TP (total protein) |
Log transformed.
|
High < control (p=0.0001), low (p=0.0012) and mid-dose groups (p<0.0001).
High dose < Na-citrate (p=0.0371). |
High <control (p=0.0330), low (p=0.0061) and mid-dose (p-=0.0013) |
|
Urea |
Log transformed.
Na-citrate > mid- (p=0.0208) and high dose (p=0.0405) groups. |
|
High dose > control (p=0.0173) and low dose (p=0.0366). |
High dose > control (p=0.0154), low dose (p=0.0261), and mid-dose (p=0.0067). |
Statistically significant differences from the pair-wise comparisons in the male animals are provided in the table below. Pair-wise comparisons were only conducted where a significant effect of group was found in the ANOVA. Results from comparisons between the control and the different aluminium citrate groups are in bold font.
MALES
Parameter |
Day 23 |
Day 64 |
Day 120 |
Day 364 |
ALB (albumin) |
|
|
|
|
ALP (alkaline phospha-tase) |
High dose > control (p=0.0268) |
High dose > control (p=0.0002), low (p=0.0002) and mid (p=0.0184) dose groups.
High dose > Na-citrate group (p<0.0001) |
|
|
ALT (alanine aminotrans-ferase) |
|
|
|
|
AST (aspartate aminotrans-ferase) |
Na-citrate>low dose (p=0.0048) |
|
|
|
A_G (albumin/ globulin ratio) |
|
High dose > control, low and mid dose groups (p<0.0001)
Mid dose > control (p=0.046).
High dose > Na-citrate group (p<0.0001)
|
Na-citrate > control (p=0.0303) |
|
CA (calcium) |
|
High dose > control, low and mid dose groups (p<0.0001)
High dose > Na-citrate group (p<0.0001) |
|
|
CHOL (cholesterol) |
|
|
|
|
CK (creatinine kinase) |
|
|
|
|
CL (chloride) |
All adjusted p-values >0.05. |
High dose < control (p=0.0003), low (p<0.0001)and mid dose (p=0.0012) groups .
High dose < Na-citrate (p=0.0073) |
|
|
CRE (creatinine) |
|
High dose > control, low and mid dose groups p<0.0001).
High dose > Na-citrate (p<0.0001) |
|
|
GLOB (globulin) |
|
High dose < control (p<0.0001), low (p<0.0001) and mid dose (p=0.0002) groups.
High dose < Na-citrate (p=0.0008) |
Na citrate < control (p=0.0003), low dose (p=0.0076), and mid-dose (p=0.0052). |
|
GLU (glucose) |
|
High dose < control (p=0.0207), low (p=0.0029) and mid dose (p=0.0136)groups |
|
|
K (potassium) |
|
|
|
|
Na (sodium) |
Na-citrate> control (p<0.0001), low dose (p=0.0006), mid-dose (p=0.0005), and high-dose (p=0.0421) groups. |
High dose < control (p=0.0247), low dose (p=0.0008), and mid-dose (p=0.0118) groups. |
|
|
Phos (phosphorus) |
|
High dose > control (p=0.0003), low (p=0.0097) and mid dose (p=0.0046)groups.
High dose > Na-citrate (p=0.0031) |
|
|
SDH (Sorbitol dehydrog-enase) |
|
|
|
|
TBIL (total bilirubin) |
|
Na-citrate <low dose, mid-dose and high dose. |
|
|
TG (triglycerides) |
|
High dose < control (p=0.0208)and low dose (p=0.0023) |
|
|
TP (total protein) |
|
High dose < control (p=0.0002), low dose (p=0.0008), and mid dose (p=0.0105) |
Na-citrate < control (p=0.0096) and low dose (p=0.0276) |
|
Urea |
|
High dose > control (p=0.0001), low dose (p<0.0001), and mid dose (p=0.0003)
Na-citrate < high dose (p<0.0001) |
|
|
In summary, significant elevations were observed predominantly in ing the high dose group relative to the other groups. Serum chemistry changes associated with aluminum toxicity such as elevated alkaline phosphatase and serum calcium were observed. The authors state the levels still remained within the normal range. Effects were most pronounced in the Day 64 cohort animals.
Tissue Metal Levels
Neonatal Pups (PND 4)
Group |
Sex |
Al µg/g, mean (sd) |
Control |
F |
0.26 (0.24) |
Low dose |
F |
0.19 (0.06) |
Mid-dose |
F |
0.41 (0.22) |
High dose |
F |
3.43 (0.21) |
Na-citrate |
F |
0.13 (0.04) |
Control |
M |
0.23 (0.15) |
Low dose |
M |
0.19 (0.08) |
Mid-dose |
M |
0.54 (0.24) |
High dose |
M |
6.72 (4.78) |
Na-citrate |
M |
0.14 (0.03) |
Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control group. This provides evidence for vertical transmission of Al to pups in-utero. There were no significant sex differences.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 300 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- No guideline Study was available on Dialuminum chloride pentahydroxide. 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 Dialuminium chloride pentahydroxide.
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. Both are GLP studies, one conducted with aluminium sulphate and the other with aluminium ammonium sulphate, which is relevant for the assessment as similar aluminium soluble salt. These studies are described below.
In the two OECD TG 416 and GLP compliant studies, aluminium sulphate (AS) and aluminium ammonium sulphate (AAS) (CAS#: 7784-25-0 as anhydrous and 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 AS study (Hirata-Koizumi et al., 2011a) include decreased drinking water consumption for both sexes in all aluminium sulphate 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 sulphate 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 was 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 bw/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 bw 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 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 anConsequentlyd 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. 100 mg/kg bw/d for DACPH as anhydrous form (molecular mass of 174.45 g/mol).
Justification for selection of Effect on developmental toxicity: via oral route:
Regarding the toxicity to reproduction, according to the specific rules for adaptation from column 1 of REACH, Annex X, 8.7.3,the two-generation reproductive toxicity studies (OECD TG 416 and initiated before 13 March 2015) are considered appropriate to address the standard information requirement, i.e. an extended One-generation reproductive toxicity study (OECD 443) basic test design (cohorts 1A and 1B without extension to include a F2 generation), one species, most appropriate route of administration, having regard to the likely route of human exposure. Therefore, based on the considerations above, it can be concluded that the overall results are likely to accurately predict the properties of the target substance (taking into account both the difference of molecular weight and Al3+ content between the substances) and are considered as adequate to fulfil the information requirement of Annex X, 8.7.
Effects on developmental toxicity
Description of key information
Combined one-year developmental and chronic neurotoxicity study (OECD 426, GLP, K, read-across, rel.1):
NOAEL (neuromuscular effects) = 30 mg (Al)/kg bw/day, i.e. for Dialuminium chloride pentahydroxide: NOAEL(neuromuscular effects) = ca. 100 mg/kg bw/day
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008-2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: OECD 426 and OECD 452
- Deviations:
- yes
- Remarks:
- : food consumption was not studied; exposure during in utero (GD 6-21) and weaning period (post-natal day (PND) 1-21), but the exposure of the rats to Al citrate continued beyond this period, until 12 months of age in one cohort
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Canada Inc.
- Age at study initiation: 14 - 16 weeks at breeding
- Weight 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
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- Group A - low dose group (mg Al/kg bw/day)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Group D - mid dose group (mg Al/kg bw/day)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Group E - high dose group (mg Al/kg bw/day)
- Dose / conc.:
- 0 other: control I
- Remarks:
- Group C - Distilled water
- Dose / conc.:
- 27.2 other: other: g/L; control II
- Remarks:
- Group B - Na citrate group (27.2 g citrate/L)
- 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.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 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. - Mortality:
- no mortality observed
- Description (incidence):
- 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 and weight changes:
- no effects observed
- Description (incidence and severity):
- 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.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Water consumption and compound intake (if drinking water study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- 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. - Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- 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. - Gross pathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- There were no statistically significant differences in gestational length between the different treatment groups.
- Dose descriptor:
- NOAEL
- Remarks:
- Maternal toxicity
- Effect level:
- 3 225 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: no treatment-related adverse effects
- Dose descriptor:
- NOAEL
- Remarks:
- Maternal toxicity
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- element
- Remarks:
- Al 3+
- Basis for effect level:
- other: no treatment-related adverse effects
- Dose descriptor:
- NOAEL
- Remarks:
- Reproductive toxicity
- Effect level:
- 3 225 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: no effects on gestational length
- Dose descriptor:
- NOAEL
- Remarks:
- Reproductive toxicity
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- element
- Remarks:
- Al 3+
- Basis for effect level:
- other: no effects on gestational length
- Abnormalities:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects: not examined.
- Remarks on result:
- not measured/tested
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The results from this study are informative for developmental and neurotoxic effects due to prenatal and chronic postnatal exposure of rats to high doses of aluminium (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.
Under the test conditions, the NOAEL for maternal toxicity is set at 300 mg Al/kg bw/day and the NOAEL for F1 generation is set at 30 mg Al/kg bw/day. - Executive summary:
This study was designed “to develop data on the potential functional and morphological hazards to the nervous system that may arise from pre-and post-natal exposure to aluminium citrate”. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions of aluminium citrate at 3 dosage levels (nominal - 30, 100 and 300 mg Al/kg bw/day. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.
Endpoints and observations in the dams included water consumption, body weight, a Functional Observational Battery (FOB), morbidity and mortality. Endpoints were assessed in both female and male pups that targeted behavioral ontogeny (motor activity, T-maze, auditory startle, the Functional Observational Battery (FOB) with domains targeting autonomic function, activity, neuromuscular function, sensimotor function, and physiological function), cognitive function (Morris swim maze), brain weight, clinical chemistry, haematology, tissue/blood levels of aluminium and neuropathology at the different dose levels and time points PND 23, 64, 120 and 364.
Statistical analyses were undertaken according to intention-to-treat, with appropriate consideration of multiple testing issues and, through the study design, also the unit of analysis. Censored analyses using survival analysis (Fixed Effects Partial Likelihood) were required for the grip strength measurements due to an equipment-defined maximum value. Females and males were analysed separately.
There were no significant Al-citrate treatment-related effects on mean body weights observed in the dams during the gestation and postnatal periods. The Na-citrate group, however, was significantly lighter than the control group on PND 15 (7.3%; p=0.0316). Eight dams in the high dose aluminium group were found to have diarrhoea compared with none in the other treatment groups. The low and mid-dose Al-citrate groups consumed more water than the control group but the high dose group did not, suggesting that the effect was not simply due to treatment. There were no significant treatment-related differences in gestational length. There were no consistent treatment-related effects observed for the FOB tests in the dams. Due to the differences in water consumption, the % of target dose differed between groups and with time through the study. In the high dose group of dams, the actual dose during the first week of gestation was 200 mg Al/kg bw/day, 67% of the target dose (300 mg Al/kg bw/day). In the last week before weaning (and sacrifice), the actual dose received by the dams was close to 175% of the target dose. Statistical analyses comparing the actual doses received by the low, mid- and high- Al-citrate treatment groups showed that the order of the dose groups was maintained, however.
The most notable treatment-related effect observed in the offspring was renal pathology – hydronephrosis, ureteral dilation, obstruction and presence of calculi - most prominently in the male pups. Higher mortality and significant morbidity were observed in the male pups in the high dose group; leading to euthanization of this group atca. study day 89. Clinical observations that showed a relationship with treatment, either directly or secondary to renal failure, were poor coat, weight loss, and haematuria. Diarrhoea was also observed. These signs were found only in the high dose Al-citrate treatment group. Haematuria was also observed in some animals in the Na-citrate group in the Day 364 cohort. Dosing with Al-citrate was associated with a reduction in body weight. The results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. In the Day 120 cohort male pups, the mid-dose animals were significantly lighter than the controls. An effect of Na-citrate was observed in the female pups in the Day 364 cohort. Overall, dosing of animals with aluminium citrate led to higher fluid consumption than in the control animals. Dosing with Na-citrate was associated with a significant increase in fluid consumption relative to that of the controls in most cohorts, with the exception of the Day 64 cohort females (fluid consumption was significantly lower in the Na-citrate group) and the Day 364 males (no significant difference between the two groups). The animals’ fluid consumption varied with time and, in mature animals, was less than expected (120 mL/kg bw/day) with implications for the actual dosage of test item received. Despite the deviations from the target dose, the low-, mid- and high-dose groups showed the required trend of lowest to highest maintaining statistically significant group differences in dose levels. For most of the study period, the actual dose received was less than the target dose in all treatment groups.
In the female pups, the mean number of days to reach vaginal opening was 31.3 (±2.1, sd) in the control group and 39.7 (±5.6, sd) in the high dose Al-citrate group, a significant difference (p<0.0001). In males, the mean number of days to reach preputial separation was 39.6 (±2.1, sd) in the control group and 42.5 (±3.2, sd) in the high dose group, also a significant difference in the pair-wise comparisons (p<0.0001). Delayed development of both male and female pups was observed in the high dose Al-citrate and Na-citrate groups. The effect is considered treatment-related but whether the effect is secondary to decreases in body weight is not clear, however.
FOB observations showed no clear treatment-related effect among the neonatal Day 364 cohort pups that were assessed at PND 5 and 11 or in the juvenile pups assessed ca.PND 22. In the adult pups, the data provide little evidence for an Al effect on the autonomic function domain, the sensimotor function domain, or excitability. Significant wasting (physiological domain), was observed in the high dose females and appears related to treatment. Characteristics of defecation (number of boluses) also showed differences with treatment. In addition, there was limited evidence of effects on activity/well-being of the pups at the high dose as reflected in fur appearance, deposits and rearing. There was some evidence for dose-response relationships between neuromuscular measurements – hind-limb and fore-limb grip strength and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. Although the FOB endpoint most consistently associated with Al-citrate treatment, grip strength, measurements showed considerably variability and a consistent ordering of the Al-treatment group responses (dose-response) was not observed at all time points. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning Day 23 cohort) or the Morris Water Maze test (Day 120 cohort).
Haematology parameters showed no significant treatment-related effects in the Day 23 cohort. In the Day 64 cohort, however, both males and females showed low grade microcytic anaemia (significantly lower mean cell volume, mean cell haemoglobin, and haematocrit). The anaemia had resolved by the end of the study in the Day 364 cohort females. Clinical chemistry results showed serum chemistry changes associated with aluminium toxicity such as elevated alkaline phosphatase and serum calcium. The authors state the levels still remained within the normal range. Effects were most pronounced in the Day 64 cohort animals. By Day 364 in the females, alkaline phosphatase levels did not differ significantly between the treatment groups.
Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control groups. There were no significant sex differences. These results suggest transfer of Al from dams to pupsin utero, although a contribution from breast milk PND 0 to 4 is also possible.Aluminium levels were assayed in several tissues in the pup cohorts.Levels of Al in whole blood were highest in the Day 23 cohort animals and declined with time, possibly due to the lower amounts of water (test solution) consumed once the pups matured. Although during the lactation period pups may have consumed some water/test solution, the results suggest that transfer of Al from dams to pups can occur through breast milk. Concentrations of Al in bone showed the strongest association with Al dose and some evidence of accumulation over time in all of the Al-treated groups. Of the central nervous system tissues, Al levels were highest in the brainstem. Although levels of Al were relatively low in the cortex (< 1µg/g), they were positively associated with Al levels in the liver and femur. In females, Al levels in the high dose group remained elevated relative to the other groups at all time points suggesting that accumulation might have occurred.
Pathological examinations showed clearly that urinary tract pathology was a treatment-related effect. The only other treatment-related effect reported on necropsy was watery, tan-coloured fluid in the digestive tract in some high dose animals, more frequently in the Day 64 group.None of the lesions seen on histopathological examination of brain tissues of the Day 364 group was treatment-related and, as these were also seen in the control group, were likely due to ageing.
This study has 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). As the offspring were dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. Urinary tract pathology was observed in rats in the high dose group, more frequently and more severe in the males. The study showed no evidence of an effect of Al-citrate on memory or learning but a more consistent effect was observed in endpoints in the neuromuscular domain.
The ambiguity as to the critical period of exposure and the time-varying water consumption complicate the derivation of a point-of-departure from this study. A LOAEL of 100 mg Al/kg bw/day for aluminium toxicity is assigned. The critical effect was a deficit in fore- and hind-limb grip strength in the mid-dose group, supported by evidence of dose response and less consistently observed effects in the mid-dose animals: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools than controls); tail pinch (mid-dose females displayed more exaggerated responses); foot-splay (mid-dose females had significantly narrower foot-splay than the controls); and the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls).
Under the test conditions, the NOAEL for maternal toxicity is set at 300 mg Al/kg bw/day and the NOAEL for F1 generation pups is set at 30 mg Al/kg bw/day for the functional hazards to the nervous system (deficit in fore- and hind-limb grip strength in the mid-dose group).
Reference
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.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 100 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
- Quality of whole database:
- No guideline Study was available on Dialuminum chloride pentahydroxide. 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 Dialuminium chloride pentahydroxide (see §"Toxicokinetics").
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.
There are 2 two-generation studies available to support a hazard assessment of the reproductive effects of aluminium. Both are GLP studies, one conducted with aluminium sulphate and the other with aluminium ammonium sulphate, which is relevant for the assessment as similar aluminium soluble salt. These studies are described below.
In the two OECD TG 416 and GLP compliant studies, aluminium sulphate (AS) and aluminium ammonium sulphate (AAS) (CAS#: 7784-25-0 as anhydrous and 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 AS study (Hirata-Koizumi et al., 2011a) include decreased drinking water consumption for both sexes in all aluminium sulphate 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 sulphate 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 was 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 bw/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 bw 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 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 anConsequentlyd 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. 100 mg/kg bw/d for DACPH as anhydrous form (molecular mass of 174.45 g/mol).
Justification for selection of Effect on developmental toxicity: via oral route:
Regarding the toxicity to reproduction, according to the specific rules for adaptation from column 1 of REACH, Annex X, 8.7.3,the two-generation reproductive toxicity studies (OECD TG 416 and initiated before 13 March 2015) are considered appropriate to address the standard information requirement, i.e. an extended One-generation reproductive toxicity study (OECD 443) basic test design (cohorts 1A and 1B without extension to include a F2 generation), one species, most appropriate route of administration, having regard to the likely route of human exposure. Therefore, based on the considerations above, it can be concluded that the overall results are likely to accurately predict the properties of the target substance (taking into account both the difference of molecular weight and Al3+ content between the substances) and are considered as adequate to fulfil the information requirement of Annex X, 8.7.
Justification for classification or non-classification
Harmonized classification:
No harmonized classification is available for the toxicity to reproduction by oral and dermal route or by inhalation of dialuminium chloride pentahydroxide.
Self classification:
Based on the available data, Dialuminium chloride pentahydroxide is not classified for the toxicity to reproduction according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Dialuminium chloride pentahydroxide is not classified for the lactation according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
