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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 242-670-9 | CAS number: 18917-91-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Link to relevant study records
- Endpoint:
- toxicity to reproduction
- Remarks:
- other: 26 wk repeated dose toxicity study
- Type of information:
- migrated information: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Acceptable, well-documented publication which meets basic scientific principles
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- other: dog
- Strain:
- other: Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Marshall Research Animals (North Rose, NY)
- Age at study initiation: 20 to 22 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually in stainless steel cages, exercised weekly
- Diet (e.g. ad libitum): Purina Certified Canine Diet #5007 + basic sodium aluminium phosphate + small amount of Mazola corn oil (0.5% w/w); 400 g of blended diet containing basic sodium aluminium phosphate was provided to each dog in a 3-hour feeding period
- Water (e.g. ad libitum): no data
- Acclimation period: ca. 5 weeks
ENVIRONMENTAL CONDITIONS
no data - Route of administration:
- oral: feed
- Vehicle:
- corn oil
- Details on exposure:
- Four-hundred grams of blended diet containing basic sodium aluminium phosphate was provided to each dog in a 3-hour feeding period.
- Details on mating procedure:
- not applicable
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test diets were blended biweekly and stored at room temperature. The concentration and homogeneity of basic sodium aluminium phosphate in each of the blended diets were determined monthly by analysis of aluminum content using atomic absorption spectrophotometry.
- Duration of treatment / exposure:
- 26 weeks
- Frequency of treatment:
- daily
- Details on study schedule:
- no mating study
- Remarks:
- Doses / Concentrations:
106, 323, 1251 mg basic Sodium aluminium phosphate/kg bw/d (females)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
112, 390, 1143 mg basic Sodium aluminium phosphate/kg bw/d (males)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
3, 10, 22, 80 mg Al/kg bw/d (females)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
4, 10, 27, 75 mg Al/kg bw/d (males)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
0, 3000, 10000 or 30000 p.p.m. basic Sodium aluminium phosphate
Basis:
nominal in diet - No. of animals per sex per dose:
- 4
- Control animals:
- yes, plain diet
- Positive control:
- no
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes :
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Basic sodium aluminium phosphate consumption was calculated from each animal's weekly food consumption and body weight. Aluminium consumption was calculated from each animal's weekly food consumption, body weight as well as the mean aluminum concentrations calculated for basal and blended diet.
FOOD EFFICIENCY: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: Yes
- Blood samples were collected for clinical laboratory analyses from all animals prior to study initiation, near midpoint and at termination. The
dogs were fasted for at least 16 hours prior to sample collection. Blood samples were collected from the jugular vein in the morning. Hematocrit, haemoglobin concentration, erythrocyte count, leukocyte count (total and differential), and platelet count were determined.
CLINICAL CHEMISTRY: Yes
- Blood samples were collected for clinical laboratory analyses from all animals prior to study initiation, near midpoint and at termination. The
dogs were fasted for at least 16 hours prior to sample collection. Blood samples were collected from the jugular vein in the morning. Serum chemistry measurements included blood urea nitrogen, creatinine, sodium, potassium, chloride, phosphorus, alanine aminotransferase, aspartate
aminotransferase, alkaline phosphatase, gamma glutamyl transferase, sorbitol dehydrogenase, total bilirubin, total protein, albumin, globulin, glucose, calcium, cholesterol, and triglyceride.
URINANALYSIS: Yes
- Urine samples were collected for clinical laboratory analyses from all animals prior to study initiation and at termination. Urine samples were collected overnight using metabolism cages.
NEUROBEHAVIOURAL EXAMINATION: No
OTHER:
- FECALANALYSIS: Yes
-- Faecal samples were collected for clinical laboratory analyses from all animals prior to study initiation and at termination. Faecal samples were collected overnight using metabolism cages. - Oestrous cyclicity (parental animals):
- not examined
- Sperm parameters (parental animals):
- Parameters examined in males:
testis weight, epididymis weight - Litter observations:
- not applicable
- Postmortem examinations (parental animals):
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Organs weighed at necropsy included heart, liver, kidneys, ovaries, testes, thyroid, adrenals, and brain. Specimens from all major organs (not mentioned in detail) were fixed by immersion in 10% neutral buffered formalin or 2.5% buffered glutaraldehyde and processed for light microscopic examination. - Postmortem examinations (offspring):
- not applicable
- Reproductive indices:
- not applicable
- Offspring viability indices:
- not applicable
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Findings were limited to high dose males.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Findings were limited to high dose males.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Findings were limited to high dose males.
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance intake: Findings were limited to high dose males. For actual ingested doses see section "Doses/concentrations"
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- effects observed, treatment-related
- Description (incidence and severity):
- Findings were limited to high dose males.
- Reproductive performance:
- not examined
- Dose descriptor:
- NOAEL
- Effect level:
- 27 mg/kg bw/day (actual dose received)
- Based on:
- element
- Remarks:
- Aluminium
- Sex:
- male
- Basis for effect level:
- other: overal effects
- Dose descriptor:
- LOAEL
- Effect level:
- 75 mg/kg bw/day (actual dose received)
- Based on:
- element
- Remarks:
- Aluminium
- Sex:
- male
- Basis for effect level:
- other: reduced body weights and food consumption with accompanying histopathological findings in liver, testes, and kidneys
- Dose descriptor:
- NOAEL
- Effect level:
- 80 mg/kg bw/day (actual dose received)
- Based on:
- element
- Remarks:
- Aluminium
- Sex:
- female
- Basis for effect level:
- other: overall effects / highest dose tested
- Dose descriptor:
- NOAEL
- Effect level:
- 390 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- basic Sodium aluminium phosphate
- Sex:
- male
- Basis for effect level:
- other: overall effects / dose corresponds to 10.000 ppm basic Sodium aluminium phosphate in diet
- Dose descriptor:
- LOAEL
- Effect level:
- 1 143 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- basic Sodium aluminium phosphate
- Sex:
- male
- Basis for effect level:
- other: reduced body weights and food consumption with accompanying histopathological findings in liver, testes, and kidneys / dose corresponds to 30.000 ppm basic Sodium aluminium phosphate in diet
- Dose descriptor:
- NOAEL
- Effect level:
- 1 251 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks:
- basic Sodium aluminium phosphate
- Sex:
- male
- Basis for effect level:
- other: overall effects / highest dose tested corresponding to 30.000 ppm basic Sodium aluminium phosphate in diet
- Clinical signs:
- not examined
- Mortality / viability:
- not examined
- Body weight and weight changes:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Histopathological findings:
- not examined
- Reproductive effects observed:
- not specified
- Conclusions:
- In this study, dietary administration of basic Sodium aluminium phosphate, to beagle dogs for 26 weeks resulted in decreased food consumption, decreased body and testis weight and histopathological changes in liver, testes and kidney of male dogs after 75 mg Al/kg bw/d. No effects were seen in females. The NOAEL was 390 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 27 mg Al/kg bw/d in male beagle dogs. The NOAEL in female beagle dogs was the highest dose tested of 1251 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 80 mg Al/kg bw/d.
- Executive summary:
In a repeated dose toxicity study comparable to OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents), male and female Beagle dogs were exposed basic Sodium aluminium phosphate at dose levels of 0, 3000, 10000 and 30000 ppm in their diets. Based on Aluminium measurements, food consumption and body weights, the male animals received 0, 112, 390 and 1143 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 4, 10, 27 and 75 mg Al/kg bw/d and the female animals 0, 106, 323 and 1251 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 3, 10, 22 and 80 mg Al/kg bw/d. In deviation to the guideline mentioned, study duration was 26 weeks and not 13 weeks. In-life observation findings were limited to a sharp, transient decrease of food consumption and a concomitant decrease of body weight in high-dose males. No effect on food consumption and body weight was observed in females. No treatment-related effects on serum chemistry, haematology or urinalysis were observed. No animal died during the study. Postmortem observations findings were limited to a decrease in testes weight in high-dose males and microscopic changes which included mild to moderate hepatocyte vacuolation accompanied by hepatocyte hypertrophy and mild bile stasis involving bile cannuliculi (three of four animals). Two high-dose males had moderate seminiferous tubule germinal epithelial cell degeneration and atrophy. According to discussion of the study authors, changes in both organs were probably a consequence of the large body weight effect observed in these animals. Other changes included very mild to mild tubular-glomerularnephritis in high-dose males. The NOAEL was 390 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 27 mg Al/kg bw/d in male beagle dogs. The NOAEL in female beagle dogs was the highest dose tested of 1251 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 80 mg Al/kg bw/d.
Reference
BODY WEIGHT AND WEIGHT GAIN and FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): There was a sharp, transient decrease of food consumption and a concomitant decrease of body weight in high-dose males. No effect on food consumption and body weight was observed in females. For actual ingested dose see section "Doses/concentrations"
FOOD EFFICIENCY: not examined
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no data
OPHTHALMOSCOPIC EXAMINATION: no data
HAEMATOLOGY, CLINICAL CHEMISTRY and URINALYSIS: No treatment-related effects on serum chemistry, haematology or urinalysis were observed.
NEUROBEHAVIOUR: not examined
ORGAN WEIGHTS and HISTOPATHOLOGY: NON-NEOPLASTIC: Findings were limited to a decrease in testes weight in high dose males and microscopic changes which included mild to moderate hepatocyte vacuolation accompanied by hepatocyte hypertrophy and mild bile stasis involving bile
cannuliculi (three of four animals). Two high-dose males had moderate seminiferous tubule germinal epithelial cell degeneration and atrophy. Changes in both organs were probably a consequence of the large body weight effect observed in these animals. Other changes included very mild to mild tubular-glomerularnephritis in high-dose males.
GROSS PATHOLOGY: no effects
HISTOPATHOLOGY: NEOPLASTIC (if applicable): not examined
HISTORICAL CONTROL DATA (if applicable): not relevant
OTHER FINDINGS
- Individual animal tissue aluminum concentrations were determined in triplicate. To provide for a conservative method and aid in statistical analysis, individual mean values were calculated by using the highest possible aluminum concentration for each determination (i.e., detection limit, limit of quantitation or actual value). These individual mean values were utilized for calculation of group mean values and for statistical analysis. Using this approach, group mean trabecular bone aluminum concentrations in males or females fed basic sodium aluminium phosphate were not significantly different from control.Similarly, brain aluminum concentrations were comparable among control and KASAL-treated males. In contrast, brain aluminum concentrations were significantly increased (p<0.05, values were 1.6 times greater than control), in high-dose (30,000 p.p.m.) females relative to control while no effect was apparent at lower KASAL dose levels.
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 294.41 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- dog
- Quality of whole database:
- All reported studies are of high quality (Klimisch score=2).
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
No reliable OECD key study isavailable on the effects of Aluminium trilactate on fertility. Data on other Aluminium compounds have been used, as it is assumed that after intake Aluminium trilactate is changed (at least in part) to ionic Aluminium and Lactate and that only ionic Aluminium is determining biological activities.
A full read-across of data based on Aluminium content and a molecular weight correction is considered for Aluminium trilactate. For detailed justification for read-across see section 5.0 justification for read across.
In a combined repeated dose / reproductive screening study (OECD guideline 422), male and female Wistar rats were exposed to Aluminium chloride, basic by oral gavage at dose levels of 40, 200 or 1000 mg/kg bw/d (equivalent to 3.6, 18 or 90 mg Al/kg bw/dl). 10 animals/sex/group were used. Males were exposed for 28 days, females between 37 to 53 days, i.e. 2 weeks prior to mating, during mating, postcoitum, and during at least 3 days of lactation. Litter was not exposed.
There were no treatment-related effects found regarding mortality, clinical signs, functional observations, organ weights, reproduction, breeding data and pup development.
Red foci on glandular mucosa of the stomach were observed in 5 of 10 males at a dose of 1000 mg/kg/d (equivalent to 90 mg Al/kg bw/d), with thickening of glandular mucosa or limiting ridge in two of these five. No other relevant abnormalities were seen. Mild to moderate subacute inflammation of the glandular mucosa and minimal to moderate superficial eosinophilic spheroids was observed in all examined animals of both sexes at 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d). No reproduction, breeding and developmental toxicity was observed for treatment up to 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d). The NOAEL for local (stomach) toxicity in males is 200 mg/kg bw/d (equivalent to 18 mg Al/kg bw/d).The LOAEL for local (stomach) toxicity in males is 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d). The NOAEL for systemic toxicity in males is 1000 mg/kg bw/d (equivalent to 90 mg Al/kg/d Al). The NOAEL for local and systemic toxicity in females is 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d).
In a repeated dose toxicity study comparable to OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents), male and female Beagle dogs were exposed basic Sodium aluminium phosphate at dose levels of 0, 3000, 10000 and 30000 ppm in their diets. Based on Aluminium measurements, food consumption and body weights, the male animals received 0, 112, 390 and 1143 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 4, 10, 27 and 75 mg Al/kg bw/d and the female animals 0, 106, 323 and 1251 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 3, 10, 22 and 80 mg Al/kg bw/d. In deviation to the guideline mentioned, study duration was 26 weeks and not 13 weeks. In-life observation findings were limited to a sharp, transient decrease of food consumption and a concomitant decrease of body weight in high-dose males. No effect on food consumption and body weight was observed in females. No treatment-related effects on serum chemistry, haematology or urinalysis were observed. No animal died during the study. Postmortem observations findings were limited to a decrease in testes weight in high-dose males and microscopic changes which included mild to moderate hepatocyte vacuolation accompanied by hepatocyte hypertrophy and mild bile stasis involving bile cannuliculi (three of four animals). Two high-dose males had moderate seminiferous tubule germinal epithelial cell degeneration and atrophy. According to discussion of the study authors, changes in both organs were probably a consequence of the large body weight effect observed in these animals. Other changes included very mild to mild tubular-glomerularnephritis in high-dose males. The NOAEL was 390 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 27 mg Al/kg bw/d in male beagle dogs. The NOAEL in female beagle dogs was the highest dose tested of 1251 mg basic Sodium aluminium phosphate/kg bw/d, corresponding to 80 mg Al/kg bw/d.
Sodium aluminium phosphate, acidic, was tested in beagle dogs in a similar protocol. No testicular effects were reported here after doses up to 88 mg Al/kg bw/d for 26 weeks (cited in EFSA (European Food Safety Authority), 2008).
DECOS (Dutch Expert Committee on Occupational Safety)(2009), EFSA (2008) and US ATSDR (United States Agency for Toxic Substances and Disease Registry)(2008) have reviewed a number of publications concerning the effects of Al on fertility. These reviews also have been taken into account for hazard assessment.
DECOS (2009) concluded that “for effects on fertility, the committee recommends not classifying metallic Aluminium, insoluble and soluble aluminium compounds due to a lack of appropriate data”.
US ATSDR (2008) has identified “anumber of oral-exposure studies examining reproductive end points in several animal species […]. In general, the results of these studies suggest that aluminum is not associated with alterations in fertility, mating success, or number of implantations, implantation losses, or litter size.
According to EFSA (2008) “studies on the reproductive toxicity in male mice (using either intraperitoneal or subcutaneous administration of aluminium nitrate or chloride) and rabbits (using gavage administration of aluminium chloride) have demonstrated the ability of aluminium to produce testicular toxicity, decreased sperm quality and reduced fertility. No reproductive toxicity was seen in females administered aluminium nitrate by gavage or dissolved in drinking water”. The table below summarises the data used by EFSA:
species |
route |
compound |
doses Al (mg/kg bw/d) |
exposure time |
Effect level |
original reference |
Sprague Dawley rats |
Drinking water |
Aluminium nitrate nonahydarate plus citric acid |
0, 50, 100 |
Females 15 days before mating and during gestation and lactation. Males were not treated |
Reduced food consumption and body weight gain (50 and 100 mg aluminium/kg bw/day).No effects on female fertility. |
Colominaet al. 2005 |
Sprague Dawley rats |
Gavage |
Aluminium nitrate nonahydarate |
0, 13, 26, 52
|
Males 60 days before mating. Females 2 weeks before mating until end of lactation |
No effects on male and female fertility. NOAEL: 52 mg Al/kg bw/day. |
Domingoet al., 1987a |
New Zealand rabbits |
Gavage |
Aluminium chloride alone / plus citric acid |
6.4 |
16 weeks |
Reduction of semen quality. NOAEL not identified |
Yousefet al. 2005 |
Beagle dogs |
Diet |
SALP, basic (KASAL) |
4, 10, 27, 75 |
26 weeks |
Reduced testes weight, epithelial germinal cells degeneration. NOAEL: 27 mg Al/kg bw/day |
Pettersenet al., 1990 |
Based on these data, the NOAEL for fertility is 27 mg Al/kg bw/d in male beagle dogs, corresponding to 294.41 mg/kg bw/d Aluminium trilactate.
References:
DECOS (Dutch Expert Committee on Occupational Safety) (2009) Aluminium and aluminium Compounds,Evaluation of the effects on reproduction, recommendation for classification. (No. 2009/02OSH, The Hague, May 28, 2009), available via internet: http://www.gezondheidsraad.nl/en/publications/aluminium-and-aluminium-compounds-evaluation-effects-reproduction-recommendation-classi#a-downloads
EFSA (European Food Safety Authority) (2008) Safety of aluminium from dietary intake, The EFSA Journal 754, 1-34, available via internet: http://www.efsa.europa.eu/de/efsajournal/pub/754.htm
US ATSDR (United States Agency for Toxic Substances and Disease Registry)(2008) Toxicological profile for Aluminium, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, Public Health Service, Agency for Toxic Substances and Disease Registry, available via internet: http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=191&tid=34
Short description of key information:
No reliable OECD key study is available on the effects of Aluminium trilactate on fertility. Data on other Aluminium compounds have been used, as it is assumed that after intake Aluminium trilactate is changed (at least in part) to ionic Aluminium and Lactate and that only ionic Aluminium is determining biological activities.
A full read-across of data based on Aluminium content and a molecular weight correction is considered for Aluminium trilactate.
Effects on fertility of Aluminium salts were assessed during studies on repeated dose toxicity or developmental toxicity. The most critical result was obtained in a study with male beagle dogs, where dietary administration of basic sodium aluminium phosphate for 26 weeks at 75 mg Al/kg bw/d resulted in decreased testicular weight and degeneration of germinal epithelium. The NOAEL in this study was 27 mg Al/kg bw/d.
Based on these data, the NOAEL for fertility is 27 mg Al/kg bw/d in male beagle dogs, corresponding to 294.41 mg/kg bw/d Aluminium trilactate.
Justification for selection of Effect on fertility via oral route:
The most critical effects on fertility were observed in this study in beagle dogs
Justification for selection of Effect on fertility via inhalation route:
no relevant route of exposure
Justification for selection of Effect on fertility via dermal route:
very low dermal absorption
Effects on developmental toxicity
Description of key information
No reliable OECD key study is available on the developmental toxicity and teratogenicity of Aluminium trilactate. Data on other Aluminium compounds have been used, as it is assumed that after intake Aluminium trilactate is changed (at least in part) to ionic Aluminium and Lactate and that only ionic Aluminium is determining biological activities.
A full read-across of data based on Aluminium content and a molecular weight correction is considered for Aluminium trilactate.
In a study on developmental toxicity of Aluminium citrate according to OECD guidelines 426 and 452 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/d). A LOAEL of 100 mg Al/kg bw/d for aluminium toxicity was identified. 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; body weight; defecation; urination; tail pinch; foot-splay; and the albumin/globulin ratio. The NOAEL in this study was 30 mg Al/kg bw/d.
In a combined repeated dose / reproductive screening study (OECD 422), 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 (equivalent to 3.6, 18 and 90 mg/Al kg bw/d) was studied. No reproduction, breeding and developmental toxicity was observed for treatment up to 90 mg Al/kg bw/d. A NOAEL for female rats was established to be 90 mg Al/kg bw/d. For males the NOAEL for local effects (stomach) was established to be 18 mg Al/kg bw/d and for systemic toxicity 90 mg Al/kg bw/d.
Based on the available data the NOAEL for developmental toxicity is 30 mg Al/kg bw/d, or recalculated to Aluminium trilactate 327.12 mg/kg bw/d.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- migrated information: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2008-2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions
- Reason / purpose 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
- Remarks:
- Doses / Concentrations:
Basis:
other: Low dose group (Group A): 30 mg Al/kg bw Mid dose group (Group D): 100 mg Al /kg bw High dose group (Group E): 300 mg Al/kg bw Control I: Distilled water Control II: Na citrate group (Group B): 27.2 g/L citrate - No. of animals per sex per dose:
- Dams: 20/group;
Offspring: 10-20 females and 10-20 males/group;
Litters: 20 litter/dose.
Beyond the treatment group allocations, dams (and their litters) were grouped according to day of delivery. This grouping allowed combining data according to postnatal day, later used in the analysis. After the end of the delivery week, litters were randomly distributed across litter groups. - Control animals:
- other: A control group received water only. A citrate control group received sodium citrate at dose equimolar to citrate in the aluminium High dose group/300 mg Al/kg/Group E - 27.2 g/L.
- Details on study design:
- Dose Selection
Doses were selected based on the results of a previous study, TEH-104 (Aluminium citrate: A 90 day toxicity study in rats. 2008. ToxTest, Alberta Research Council, Report No.: TEH-104) and the maximum solubility of aluminium citrate in water (high dose). The number of dose levels and dose spacing was according to guideline.
Dams & Sires
Allocation to Treatment Groups
Rats were randomly allocated to treatment groups and randomly selected for breeding using the SAS PROC PLAN procedure.
Allocation to Shelf/Rack
Prior to breeding, a Youden square was used to produce equal representation of the treatment groups within each shelf of the rack.
Location of the breeding pairs was also dictated using a Youden square. After insemination, each dam was returned to her original cage and remained at that location until postnatal day 1 or euthanasia.
As the proportion of dams in each treatment groups that would deliver on a specific day could not be predicted, extra breeding pairs were included in the study. After the end of the week during which deliveries were expected, litters that were eligible to enter the study (≥4 pups of each sex) were randomly chosen to provide a balanced distribution of litters per treatment group per delivery day.
Pups
Litter Normalisation
At PND 4, litters were normalized to 4 males and 4 females using random numbers. Of the extra pups, 4 males and 4 females per treatment group were randomly chosen for whole body aluminium, iron, manganese, copper and zinc assay.
Allocation to Cohort
Also on PND 4, one pup per sex and normalised litter was assigned by number to each of 4 cohorts (Cohort 1- PND1- 22, Cohort 2 – PND 23-64, Cohort 3- PND 65- 120, and Cohort 4 – PND 121- 364) associated with observations, examinations and sacrifice.
In addition to treatment group allocations, dams (and their litters) were also grouped according to day of delivery to facilitate scheduling of the different procedures.
Allocation to Shelf/Rack
Pups were weaned at PND 22 by moving them to individual ventilated caging using another Youden square to determine their distribution within the rack.
Blinding
Assessors were blinded to treatment group. Treatment groups were identified with letters - Group A (30 mg Al/kg bw/day, Low dose group), Group B (Na citrate group), Group C (Control group), Group D (100 mg Al/kg bw/day, Mid dose group), and Group E (300 mg Al/kg bw/day, High dose group). Dams and sires were identified by ear tags 3 days after arrival at the facility. Pups were identified on PND 4 within micro tattoo on the feet, and on PND 21 (at weaning) with an ear tag. Cages were identified by cage cards. - Maternal examinations:
- Parental animals
Dams
Morbidity and Mortality
All dams underwent daily morbidity and mortality checks and a clinical examination was performed on the day of delivery.
Functional Observational Battery (FOB)
Schedule: Gestational days (GD) 7 and 13 and on postnatal days (PND) 3 and 10.
Content: The FOB (adults) included:
- cage-side assessment,
- handling assessment,
- open field observations (posture, involuntary movements, abnormal motor movements), and
- sensory and neuromuscular observations:
- footsplay and
- fore-limb grip strength and
- hind-limb grip strength.
Body weights
Schedule: GD 6, 13, and 20, PND 1, 8, 15, and 22.
Body weight on PND 1 was examined but not included in the analysis.
Water consumption
Schedule: GD 6, 13, 20, and then on PND 1, 8, 15, and 22. - Ovaries and uterine content:
- No data.
- Fetal examinations:
- Not applicable.
- Statistics:
- See "any other information on materials and methods incl. tables"
- Indices:
- No data.
- Historical control data:
- No data.
- Details on maternal toxic effects:
- Maternal toxic effects:no effects
Details on maternal toxic effects:
Mortality
No mortality was observed in the dams during the gestation and postnatal periods in the control group, the low-dose group, the mid-dose group or the high-dose group; 20 dams were euthanized on the scheduled dates in each group. One dam that stopped nursing was euthanized early in the sodium citrate group.
Body weight
The ANOVA showed a significant effect of group (p=0.021). This was due to lower body weights in the sodium citrate group. At PND15, the mean weight of the Na-citrate group was 7.3% less than in the controls. There were no significant differences in mean body weights in dams between the aluminium-treated groups and the control group during the gestational and postnatal period.
Gestation Length
There were no statistically significant differences in gestational length between the different treatment groups.
Clinical Observations
All dams underwent daily morbidity and mortality checks during the gestational period and a clinical examination was performed on the day of delivery. Abnormal clinical observations were reported for only one dam during the gestational period.
During the postnatal period, 4 animals in the control group, 8 in the Na-citrate group, 4 in the low-dose group, 6 in the mid-dose group, and 12 in the high dose group exhibited clinical signs. Most signs were considered mild, for example alopecia and porphyrin staining. Slight dehydration was noted in 4 dams in the Na-citrate group. Diarrhoea was reported in 8 dams in the high dose aluminium group only, and thus appears to be a treatment-related effect.
Water Consumption
The table below the ranges of mean fluid consumption in mL/day (mL/kg bw/day) for the different groups for the gestation and lactation periods:
Group/Period Gestation Lactation
Control 23.0 to 31.5 (67 to 79) 35.1 to 60.6 (99 to 179)
Low Dose 35.9 to 43.7 (103 to 108) 40.1 to 60.9 (114 to 177)
Mid-Dose 42.0 to 45.2 (112 to 123) 40.9 to 69.0 (136 to 201)
High-Dose 27.4 to 31.3 (78 to 80) 39.7 to 70.2 (120 to 211)
Na-citrate 26.2 to 29.3 (66 to 76) 35.1 to 68.0 (106 to 213)
A significant effect of group was found in the ANOVA (p<0.0001). Pairwise between-group comparisons showed that the low dose group consumed significantly more water than the sodium citrate (p=0.011) and water control (p=0.0028) groups. The mid-dose group consumed significantly more than the sodium citrate (p<0.0001), water control (p<0.0001) and high dose groups (p=0.023). The differences were most marked during the gestation period.
As increased water consumption was not observed in the high dose group, the effect is not likely due to treatment.
Daily Al dosage
The target dose for the low dose group was 30 mg Al/kg bw/day, for the mid-dose 100 mg Al/kg bw/day and for the high dose 300 mg Al/kg bw/day.
Despite the deviations from the target dose, the low, medium and high dose groups showed the required trend of lowest to highest maintaining group differences in dosage.
FOB
During the gestation period, approach response, arousal, bizarre behaviour, circling, clonic convulsions, clonic convulsions rating, gait, posture, pupil response, pupil size, startle, stereotypic behaviour, tail pinch, tonic convulsions, tonic convulsions rating, total gait, tremors, tremors rating, vocalization, and writhing were zero for all dams.
The group effect (repeated measures ANOVA) for defecation (p=0.052), rearing (p=0.344), urination (p=0.487) and foot splay (p=0.089) did not reach statistical significance. A significant group effect was observed for hind limb grip strength (p=0.0047; censored analysis) driven by a lower grip strength in the Na-citrate group compared to the low and high dose groups.
During the postnatal period, bizarre behavior, circling, clonic convulsions, clonic convulsions rating, gait, posture, pupil response, stereotypic behavior, tonic convulsions, tonic convulsions rating, total gait, tremors, tremors rating, and writhing were zero for all dams.
The group effect (repeated measures ANOVA) for approach response (p=0.518), arousal (p=0.146), defecation (p=0.096), pupil size (p=0.413), rearing (p=0.151), startle (p=0.668), tail pinch (p=0.242), urination (p=0.793), vocalization (p=0.092), and foot splay (p=0.142) did not reach statistical significance. A significant across groups difference (censored analysis) was observed for forelimb grip strength (p=0.0031). Pair-wise comparisons showed that the mid-dose group was significantly less than the sodium citrate group (p=0.0005) and the high dose group (p=0.0115). The low dose group was significantly less than the sodium citrate group (p=0.012) and the control group was significantly less than the sodium citrate group (p=0.0076). The group effect for hind limb grip strength did not reach statistical significance (p=0.073) so pair-wise comparisons were not conducted.
Overall, there was no consistent effect of treatment group on any of the FOB characteristics in the dams. - Dose descriptor:
- NOAEL
- Effect level:
- 3 225 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day
- Based on:
- element
- Remarks:
- Al 3+
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:not examined
Details on embryotoxic / teratogenic effects:
Not applicable. - Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The results from this study are informative for developmental and neurotoxic effects due to prenatal and chronic postnatal exposure of rats to high doses of aluminium (30 mg Al/kg bw/day, 100 mg Al/kg bw/day and 300 mg Al/kg bw/day). As the F1 generation was dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. This does not affect the formal reliability of the study.
The results in the Day 364 cohort show a clear, consistent effect on post-weaning body weight in the high dose Al-citrate group in both male and female pups. An effect of Na-citrate was observed in the female pups. Urinary tract pathology was observed in high dose rats, more frequently in the males. The results showed no evidence of an effect on memory or learning. Fairly consistent results were observed for the critical effect, fore- and hind-limb grip strength, and this was supported by the following less consistently observed effects also observed in the mid-dose (100 mg Al/kg bw/day) group: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools that controls); tail pinch (mid-dose females displayed more exaggerated responses); foot splay (mid-dose females had significantly narrower foot splay than the controls); the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls). No treatment-related differences in FOB characteristics were observed in the neonatal and juvenile pups. A LOAEL of 100 mg Al/kg bw/day for aluminium repeated dose toxicity is assigned based on this study.
Delayed sexual maturation, measured as delayed vaginal opening in females and delayed preputial separation in males, was observed in the high dose Al-citrate group of this study. The same effect, although somewhat less pronounced, was also seen in the sodium citrate control group. Based on the observed upward deviations from the target dose in the Al citrate groups and the data on water consumption seen in the first weeks after weaning, it is possible that both in the pre- and post-weaning stage, the animals in the Al citrate groups received considerably more citrate than the sodium citrate control group. Moreover, the calculated Al dose during the immediate post-weaning period was more than twice the target dose, which may have contributed to post-natal systemic toxicity due to exposure to the test substance. Given that effects were seen in both the Al-citrate high-dose group and the NA-citrate group, no Al-based LOAEL/NOAEL can be suggested based on the sexual maturation results in this study.
Body weight differences at end-of-weaning, relative to controls, occurred in the high-dose Al-citrate group as well as in the sodium citrate group and are considered to be treatment-related but the role of Al is unclear. The relative differences between the high-dose Al-citrate group and the sodium citrate group may be related to differences in liquid consumption. - Executive summary:
This study was designed “to develop data on the potential functional and morphological hazards to the nervous system that may arise from pre- and post-natal exposure to aluminium citrate”. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions of aluminium citrate at 3 dosage levels (nominal - 30, 100 and 300 mg Al/kg bw/day. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al citrate and Na-citrate were administered to damsad libitumviadrinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.
Endpoints and observations in the dams included water consumption, body weight, a Functional Observational Battery (FOB), morbidity and mortality. Endpoints were assessed in both female and male pups that targeted behavioral ontogeny (motor activity, T-maze, auditory startle, the Functional Observational Battery (FOB) with domains targeting autonomic function, activity, neuromuscular function, sensimotor function, and physiological function), cognitive function (Morris swim maze), brain weight, clinical chemistry, haematology, tissue/blood levels of aluminium and neuropathology at the different dose levels and time points PND 23, 64, 120 and 364.
Statistical analyses were undertaken according to intention-to-treat, with appropriate consideration of multiple testing issues and, through the study design, also the unit of analysis. Censored analyses using survival analysis (Fixed Effects Partial Likelihood) were required for the grip strength measurements due to an equipment-defined maximum value. Females and males were analysed separately.
There were no significant Al-citrate treatment-related effects on mean body weights observed in the dams during the gestation and postnatal periods. The Na-citrate group, however, was significantly lighter than the control group on PND 15 (7.3%; p=0.0316). Eight dams in the high dose aluminium group were found to have diarrhoea compared with none in the other treatment groups. The low and mid-dose Al-citrate groups consumed more water than the control group but the high dose group did not, suggesting that the effect was not simply due to treatment. There were no significant treatment-related differences in gestational length. There were no consistent treatment-related effects observed for the FOB tests in the dams. Due to the differences in water consumption, the % of target dose differed between groups and with time through the study. In the high dose group of dams, the actual dose during the first week of gestation was 200 mg Al/kg bw/day, 67% of the target dose (300 mg Al/kg bw/day). In the last week before weaning (and sacrifice), the actual dose received by the dams was close to 175% of the target dose. Statistical analyses comparing the actual doses received by the low, mid- and high- Al-citrate treatment groups showed that the order of the dose groups was maintained, however.
The most notable treatment-related effect observed in the offspring was renal pathology – hydronephrosis, ureteral dilation, obstruction and presence of calculi - most prominently in the male pups. Higher mortality and significant morbidity were observed in the male pups in the high dose group; leading to euthanization of this group 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 pupsin utero, although a contribution from breast milk PND 0 to 4 is also possible.Aluminium levels were assayed in several tissues in the pup cohorts.Levels of Al in whole blood were highest in the Day 23 cohort animals and declined with time, possibly due to the lower amounts of water (test solution) consumed once the pups matured. Although during the lactation period pups may have consumed some water/test solution, the results suggest that transfer of Al from dams to pups can occur through breast milk. Concentrations of Al in bone showed the strongest association with Al dose and some evidence of accumulation over time in all of the Al-treated groups. Of the central nervous system tissues, Al levels were highest in the brainstem. Although levels of Al were relatively low in the cortex (< 1µg/g), they were positively associated with Al levels in the liver and femur. In females, Al levels in the high dose group remained elevated relative to the other groups at all time points suggesting that accumulation might have occurred.
Pathological examinations showed clearly that urinary tract pathology was a treatment-related effect. The only other treatment-related effect reported on necropsy was watery, tan-coloured fluid in the digestive tract in some high dose animals, more frequently in the Day 64 group.None of the lesions seen on histopathological examination of brain tissues of the Day 364 group was treatment-related and, as these were also seen in the control group, were likely due to ageing.
This study has many strengths. It was conducted according to GLP with a design based on OECD TG #426. The study used adequate numbers of animals and randomization to reduce bias, assessed endpoints in both female and male offspring, and studied a wide range of neurotoxicity endpoints. Haematology, clinical chemistry, pathology and general toxicity endpoints were also assessed. Three dose levels were used although the highest was close to the MTD.Although representative of actual human exposures, extending the period of exposure beyond weaning until day 364 leads to ambiguity in interpretation of the results as effects observed later in the study may have resulted from either later exposures or exposures during periods critical for development. There were a number of deviations from protocol that are clearly described in the study report. The report mentions that the observation protocol was not always consistently followed and that the clinical observations for the Group B and E animals started on April 3, 2008 instead of March 19, 2008. Some auditory startle data was lost due to an equipment malfunction (16 animals in Cohort 1 and 7 animals in Cohort 2) resulting in a “loss of statistical power for this part of the study”. Some necropsies were not completed “22 pre-weaning animals (eight from one litter) and 3 post-weaning animals that died or were euthanized prior to scheduled sacrifice did not have a necropsy completed”. The equipment used to measure grip strength had a maximum capacity of 700g leading to the possibility of underestimation of grip strength in larger animals, particularly in the males. Overall, these deviations were unlikely to have impacted the results of the study.
The results from this study are informative for developmental and neurotoxic effects due to combined prenatal and chronic postnatal exposure of rats to high doses of aluminium (30 mg Al/kg bw/day, 100 mg Al/kg bw/day and 300 mg Al/kg bw/day).Asthe offspring were dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. Urinary tract pathology was observed in rats in the high dose group, more frequently and more severe in the males. The study showed no evidence of an effect of Al-citrate on memory or learning but a more consistent effect was observed in endpoints in the neuromuscular domain.
The ambiguity as to the critical period of exposure and the time-varying water consumption complicate the derivation of a point-of-departure from this study. A LOAEL of 100 mg Al/kg bw/day for aluminium toxicity is assigned. The critical effect was a deficit in fore- and hind-limb grip strength in the mid-dose group, supported by evidence of dose response and less consistently observed effects in the mid-dose animals: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools than controls); tail pinch (mid-dose females displayed more exaggerated responses); foot-splay (mid-dose females had significantly narrower foot-splay than the controls); and the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls).
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:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 327.12 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- All reported studies are of high quality (Klimisch score=2).
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
No reliable OECD key study isavailable on the developmental toxicity and teratogenicity of Aluminium trilactate. Data on other Aluminium compounds have been used, as it is assumed that after intake Aluminium trilactate is changed (at least in part) to ionic Aluminium and Lactate and that only ionic Aluminium is determining biological activities.
A full read-across of data based on Aluminium content and a molecular weight correction is considered for Aluminium trilactate. For detailed justification for read-across see section 5.0 justification for read across.
A study on developmental toxicity of Aluminium citrate according to OECD guidelines 426 and 452 is available:
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/d). Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al citrate and Na-citrate were administered to damsad libitumvia 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 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.
There were no significant Al-citrate treatment-related effects on mean body weights observed in the dams during the gestation and postnatal periods. There were no significant treatment-related differences in gestational length.
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 euthanisation of this group atca. 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/d) with implications for the actual dosage of test item received. Despite the deviations from the target dose, the low-, mid- and high-dose groups showed the required trend of lowest to highest maintaining statistically significant group differences in dose levels. For most of the study period, the actual dose received was less than the target dose in all treatment groups.
In the female pups, the mean number of days to reach vaginal opening was 31.3 (±2.1, sd) in the control group and 39.7 (±5.6, sd) in the high dose Al-citrate group, a significant difference (p<0.0001). In males, the mean number of days to reach preputial separation was 39.6 (±2.1, sd) in the control group and 42.5 (±3.2, sd) in the high dose group, also a significant difference in the pair-wise comparisons (p<0.0001). Delayed development of both male and female pups was observed in the high dose Al-citrate and Na-citrate groups. The effect is considered treatment-related but whether the effect is secondary to decreases in body weight is not clear, however.
FOB observations showed no clear treatment-related effect among the neonatal Day 364 cohort pups that were assessed at PND 5 and 11 or in the juvenile pups assessedca.PND 22. In the adult pups, the data provide little evidence for an Al effect on the autonomic function domain, the sensimotor function domain, or excitability. Significant wasting (physiological domain), was observed in the high dose females and appears related to treatment. Characteristics of defecation (number of boluses) also showed differences with treatment. In addition, there was limited evidence of effects on activity/well-being of the pups at the high dose as reflected in fur appearance, deposits and rearing. There was some evidence for dose-response relationships between neuromuscular measurements – hind-limb and fore-limb grip strength and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. Although the FOB endpoint most consistently associated with Al-citrate treatment, grip strength, measurements showed considerably variability and a consistent ordering of the Al-treatment group responses (dose-response) was not observed at all time points. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning Day 23 cohort) or the Morris Water Maze test (Day 120 cohort).
Haematology parameters showed no significant treatment-related effects in the Day 23 cohort. In the Day 64 cohort, however, both males and females showed low grade microcytic anaemia (significantly lower mean cell volume, mean cell haemoglobin, and haematocrit). The anaemia had resolved by the end of the study in the Day 364 cohort females. Clinical chemistry results showed serum chemistry changes associated with aluminium toxicity such as elevated alkaline phosphatase and serum calcium. The authors state the levels still remained within the normal range. Effects were most pronounced in the Day 64 cohort animals. By Day 364 in the females, alkaline phosphatase levels did not differ significantly between the treatment groups.
Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control groups. There were no significant sex differences. These results suggest transfer of Al from dams to pupsin utero, although a contribution from breast milk PND 0 to 4 is also possible. Al 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 (< 1 µg/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.
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, 100 and 300 mg Al/kg bw/d). 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/d 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).
In a combined repeated dose / reproductive screening study (OECD 422), 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 (equivalent to 3.6, 18 and 90 mg/Al kg bw/d) was studied.
Mild to moderate subacute inflammation of the glandular mucosa and minimal to moderate superficial eosinophilic spheroids in all examined animals of both sexes at 1000 mg/kg bw/d .
Red foci on glandular mucosa of the stomach were observed in 5 of 10 males at a dose of 1000 mg/kg/d (equivalent to 90 mg Al/kg bw/d), with thickening of glandular mucosa or limiting ridge in two of these five. No other relevant abnormalities were seen. Mild to moderate subacute inflammation of the glandular mucosa and minimal to moderate superficial eosinophilic spheroids was observed in all examined animals of both sexes at 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d).
No reproduction, breeding and developmental toxicity was observed for treatment up to 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d).
Therefore, the overall NOAEL for female rats was established to be 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d). For males the NOAEL for local effects was established to be 200 mg/kg bw/d (equivalent to 18 mg Al/kg bw/d) and for systemic toxicity 1000 mg/kg bw/d (equivalent to 90 mg Al/kg bw/d).
DECOS(Dutch Expert Committee on Occupational Safety)(2009), EFSA (European Food Safety Authority) (2008),Krewski, et al. (2007).US ATSDR (United States Agency for Toxic Substances and Disease Registry)(2008),WHO (World Health Organisation) (2003) and WHO IPCS EHC (World Health Organisation International Programme on Chemical Safety Environmental Health Criteria)(1997) have reviewed several studies on the effects of Aluminium trilactate and other Aluminium salts to pre-natal development. No effects were observed on pregnancy rate, implantations, resorptions, and live and dead fetuses.
No homogeneous results were obtained from those studies. In some studies skeletal abnormalities and reduction of fetal body weights (decreased or retarded ossification) are reported, while not in others. Also the effect levels are ambiguous.
DECOS(2009) concluded that no effects on reproductive parameters (pregnancy rate, absorptions, implantation sites, litter size, and pup weight at birth) were seen at dose levels that did not induce general toxic effects, whereas in fetuses of dams orally treated with soluble aluminium compounds at dose levels inducing general toxicity, decreased fetal weights and retarded ossification were observed.
But especially neurodevelopmental and behavioural effects were also observed after oral administration of soluble aluminium compounds at concentrations that did not induce general toxic effects in the dams.
EFSA (2008) came to the conclusion that “high doses of aluminium nitrate, chloride or lactate given by gavage were able to induce some signs of embryotoxicity in mice and rats, in particular, reduced fetal body weight or pup weight at birth and delayed ossification. […] After dietary exposure of rats to aluminium chloride and lactate the lowest NOAEL was 100 mg aluminium/kg bw/day, respectively. Gavage administration of aluminium hydroxide at doses providing up to 264 mg aluminium/kg bw/day was without embryotoxic effects in rats”.
Reduced pup body weight was reported in some studies at Aluminium levels as low as 13 mg Al/mg bw/d (studies reviewed by EFSA, 2008; USATSDR, 2008). US ATSDR (2008) states, that ”since some of these studies did not report the aluminum content of the basal diet, their usefulness in establishing dose-response relationships is limited.” Moreover “most studies have shown that aluminum does not adversely affect birth weight in the absence of effects on maternal body weight” (USATSDR, 2008).
According to US ATSDR (2008) “developmental toxicity studies in animals have shown that oral gestational exposure to aluminum induced skeletal variations such as delayed ossification in rats and mice under conditions that enhanced its uptake, particularly maternal intake of compounds that are highly bioavailable (e.g., aluminum citrate and nitrate), concurrent exposure to dietary constituents that contribute to increased absorption of aluminum (e.g., citrate), and/or bolus administration by gavage”. And “because the developmental effects of orally administered aluminum appear to be dependent on the bioavailability of the form in which it is administered and the presence of dietary components that promote aluminum uptake, additional information on compound-related differences in aluminum uptake and effectiveness during pregnancy and postnatal development would help in assessing the relevance of the animal data to oral exposures in humans. For example, gavage administration of low doses of aluminum (38–77 mg Al/kg/day) as aluminum nitrate during gestation induced skeletal variations in rats (Paternain et al. 1988), indicating that the LOAEL for this effect is below the neurotoxicity NOAEL of 62 mg Al/kg/day for aluminum lactate in adult mice used to derive the MRL [= minimal risk level, remark by submitter]. The Paternain et al. (1988) LOAEL was not considered to be appropriate for MRL consideration due to concern that gavage does not realistically represent environmental aluminum intake (i.e., the LOAEL could be unnaturally low compared to dietary exposure because the skeletal effects could be related to phosphate binding caused by the bolus administration), and that nitrate represents an unusually bioavailable form of aluminum.”
WHO (2003) summarised: “The developmental toxicity of aluminium by the oral route is highly dependent on the form of aluminium and the presence of organic chelators that influence bioavailability. Aluminium hydroxide did not produce either maternal or developmental toxicity when it was administered by gavage during embryogenesis to mice at doses up to 92 mg of aluminium per kg of body weight per day (Domingo et al., 1989) or to rats at doses up to 265 mg of aluminium per kg of body weight per day (Gomez et al., 1990). When aluminium hydroxide at a dose of 104 mg of aluminium per kg of body weight per day was administered with ascorbic acid to mice, no maternal or developmental toxicity was seen, in spite of elevated maternal placenta and kidney concentrations of aluminium (Colomina et al., 1994); on the other hand, aluminium hydroxide at a dose of 133 mg of aluminium per kg of body weight per day administered with citric acid produced maternal and fetal toxicity in rats (Gomez et al., 1991). Aluminium hydroxide (57 mg of aluminium per kg of body weight) given with lactic acid (570 mg/kg of body weight) to mice by gavage was not toxic, but aluminium
lactate (57 mg of aluminium per kg of body weight) produced developmental toxicity, including poor ossification, skeletal variations, and cleft palate (Colomina et al., 1992).”
Most sensitive effects were postnatal FOB findings (deficits in fore- and hind-limb grip strength) in the oral study according to OECD guidelines 426 and 452 on rats, which was not considered in the mentioned assessment reports due to late finalisation.Based on these data the NOAEL for developmental toxicity is 30 mg Al/kg bw/d, or recalculated to Aluminium trilactate327.12 mg/kg bw/d.
References:
DECOS (Dutch Expert Committee on Occupational Safety) (2009) Aluminium and aluminium Compounds,Evaluation of the effects on reproduction, recommendation for classification. (No. 2009/02OSH, The Hague, May 28, 2009), available via internet: http://www.gezondheidsraad.nl/en/publications/aluminium-and-aluminium-compounds-evaluation-effects-reproduction-recommendation-classi#a-downloads
EFSA (European Food Safety Authority) (2008) Safety of aluminium from dietary intake, The EFSA Journal 754, 1-34, available via internet: http://www.efsa.europa.eu/de/efsajournal/pub/754.htm
Krewski, et al. (2007). Human Health Risk Assessment for Aluminium, Aluminium Oxide, and Aluminium Hydroxide, A Report Submitted to the US Environmental Protection Agency. J Toxicol Environ Health B Crit Rev. 10 Suppl 1:1-269. Available via internet: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782734/
US ATSDR (United States Agency for Toxic Substances and Disease Registry)(2008) Toxicological profile for Aluminium, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, Public Health Service, Agency for Toxic Substances and Disease Registry, available via internet: http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=191&tid=34
WHO (World Health Organisation) (2003)Background document for development of WHOGuidelines for Drinking-water Quality, Guidelines for drinking-water quality, 2nd ed. Addendum to Vol. 2.Health criteria and other supporting information.World Health Organization, Geneva, 1998. Available via internet: http://www.who.int/water_sanitation_health/dwq/chemicals/en/aluminium.pdf
WHO IPCS EHC (World Health Organisation International Programme on Chemical Safety Environmental Health Criteria)(1997) Aluminium (Environmental health criteria; 194), IPCS, World Health Organization, Geneva, available via internet: http://www.inchem.org/documents/ehc/ehc/ehc194.htm
Justification for selection of Effect on developmental toxicity: via oral route:
The most sensitive effects observed for development were postnatal FOB findings (deficits in fore- and hind-limb grip strength) in the oral study according to OECD guidelines 426 and 452 in rats.
Justification for selection of Effect on developmental toxicity: via inhalation route:
no relevant route of exposure
Justification for selection of Effect on developmental toxicity: via dermal route:
very low dermal absorption
Justification for classification or non-classification
In conclusion, the results of the available data on toxicity to reproduction, developmental toxicity and teratogenicity indicate that Aluminium trilactate, does not need to be classified for toxicity to reproduction, developmental toxicity and teratogenicity according to Directive 67/548/EEC as well as CLP, EU GHS (Regulation 1272/2008/EC) and therefore labelling is not necessary.
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.
