Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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 (Spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured
Qualifier:
equivalent or similar to guideline
Guideline:
other: Japanese guidelines for the "designation of food additives and for the revision of standards for the use of food additives".
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: (P) 5 wks; (F1) 3 wks
- Housing: animals were housed individually (except during the acclimation, mating and nursing periods) in suspended wire-mesh cages. From day 17 of gestation to day 21 after delivery, the wire-mesh floor of the cage was replaced with a stainless-steel tray, and individual dams and litters were reared using wood chips as bedding.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet: standard rat diet (CRF-1;Oriental YeastCo.,Ltd.,Tokyo,Japan), ad libitum
- Water: ion-exchanged drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 36-59
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared by dissolving the test item in ion-exchanged water, and served as drinking water to the animals. The dosing solution were prepared at least every 6 days and kept under cool conditions until serving and drinking solutions were replaced at least once every 4 days.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until successful copulation occurred or the mating period of 2 weeks had elapsed.
- After 14days of unsuccessful pairing replacement of first male by another male from the same group with proven fertility (who had been proved to copulate with limits of not less than 7 days)
- Proof of pregnancy: vaginal plug and/ sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the study the, the concentrations of test material in drinking water were analyzed in the first and last preparations and once every 3 months, and confirmed to be 97.5 – 106.3% of the target high performance liquid chromatography. As contained in the drinking water for the control group was less than the quantitation limit (5 µg/mL).
Duration of treatment / exposure:
P males: 10 weeks prior to mating, during mating and up to parturition of the paired females
P females: 10 weeks prior to mating, during mating and during lactation until sacrifice after weaning of their pups (PND26)
F1: selected on PND 21-25 (designated as day 0 of dosing) and were treated in same way as P males and females
Frequency of treatment:
daily, continuously
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
- Age at mating of the mated animals in the study: 13 weeks (F1), 15 weeks (P)
Remarks:
Doses / Concentrations:
120, 600, 3000 ppm
Basis:
nominal in water
for actual doses receive see table 1 under any other information on materials and methods including tables
No. of animals per sex per dose:
24 P males and females
24 F1 males and females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on the results of a foregoing range finding study, in which animals were given AS dissolved in drinking water at 1000, 3000, 10,000 or 30,000 ppm. Males were dosed for 7 weeks, beginning 14 days before mating to day 4 of lactation throughout the mating and gestation period. The following adverse effects were observed: marked decreased in body weight as a result of water avoidance at the highest dose group; decrease of water consumption, in all treatment group; decreased food consumption and body weight at 3000 ppm and above; thickening of the limiting ridge in the stomach, and atrophy of the thymus and spleen at 10,000 ppm; the relative weights of the liver, thymus and spleen were decreased in females in 3000 and 10,000 ppm groups. Therefore 120, 600 or 3000 ppm were selected as the dose levels for the main study.
- Rationale for animal assignment: by stratified random sampling based on body weight

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, for Dams body weight was recorded on gestational days 0, 7, 14 and 20 and days 0, 7, 14 and 21 of lactation (and additional day 4 of lactation for body weight)

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Time schedule for examinations: weekly. For dams, food consumption was recorded on gestational days 0, 7, 14 and 20 of gestation and days 0, 7, 14 and 20 of lactation (and additional day 4 of lactation for body weight)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice a week and on days 0, 7, 14 and 20 of gestation and days 0, 4, 7, 11, 14, 17, 19 and 21 of lactation
Oestrous cyclicity (parental animals):
Daily vaginal lavage samples were evaluated for each female for estrous cyclicity throughout the last 2 weeks of the premating period and during cohabitation until evidence of copulation was detected. Females with repeated 4-6 day estrous cycles were considered as having normal estrous cycles.
Sperm parameters (parental animals):
Sperm parameters were determined in all P and F1 adult males on day of sacrifice; right testis was used to count testicular homogenization-resistant spermatid heads; the right epididymal cauda was weighed and used for sperm analysis.
For sperm motility, the percentage of motile sperm and progressively motile sperm and the swimming speed and pattern were determined using a somputer-assissted cell motion analyzer (TOX IVOS). After recording sperm motion, the cauda epididymal fluid was diluted and the sperm were enumerated with a hematocytometer und a light microscope. Sperm count per gram of epididymal tissue was obtained by dividing the total count by the gram weight of the caudal epididymis.
Sperm morphology was studied by staining sperm with eosin and mount it on glass slides. 200 sperm in each sample were examined under light microscope and percentage of morphologically abnormal sperm was calculated.
Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible, no adjustment was made for litters of fewer than eight pups); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
Number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities. Clinical signs of toxicity (daily) and the body weight of live pups were measured on PND 0, 4, 7, 14 and 21.

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

OTHER:
- developmental landmarks: Pinna unfolding in all F1 and F2 pups (PND1 - PND4); anogenital distance (AGD) measured on PND4 in all F1 and F2 pups; incisor eruption for one male and one female F1 and F2 pup selected from each litter were evaluated on PND8 and eye opening on PND12 until each pup fulfilled criteria.
- neuromotor performances: surface righting reflex, negative geotaxis and midair righting reflex were assessed on PND5, 8 and 18 for one male and female F1 and F2 pup selected from litter.
- neurobehavioral examinations:
locomotor activity - 10 male and 10 female F1 rats randomly selected from each group at 4 weeks of age (multi-channel activity monitoring system used).
T-maze test - water-filled multiple T-maze test was conducted in 10 male and 10 female F1 rats selected from each group at 6 weeks of age.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced (after parturition of their paired females).
- Maternal animals: All surviving animals, after the last litter of each generation were weaned, on PND26.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
The number of uterine implantation sites was recorded for each dam. The testis and epididymis were prepared for microscopic examination and weighed. The brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididymis, seminal vesicles (with coagulating gland and their fluids), ventral prostate, uterus and ovaries in males and females were weighed before fixation, fixed and underwent macroscopic examination. The thyroid and seminal vesicles were weighed after fixation. In 10 F1 females, randomly selected from the control and highest dose group, the number of primordial follicles was counted in about 40 sections per ovary.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathologic evaluations were performed:
- in all animals of the control and the highest dose group
- in females with abnormal estrous cycle, abnormal delivery or total dead pups
- in males and females without evidence of copulation or insemination
- in all animals with grossly abnormal reproductive organs
- testes and epididymis were fixed in Bouin's solution and preserved in 70% ethanol; all other organs were fixed in 10% neutral bufferes formalin.

Testes, epididymis, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina were sectioned, stained with hematoxylin-eosin and examined under a light microscope. When treatment-related changes were found in the highest dose group, the same tissue from the next lower dose group then were examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 26 days of age.
- all pups found dead before weaning were necropsied immediately, following the adjustment of litter size on PND4, culled pups were sacrificed by carbon monoxide and subjected to gross external and internal examination

HISTOPATHOLOGY / ORGAN WEIGTHS
For one male and one female F1 and F2 weanlings selected from each dam:
- the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries were removed and prepared for microscopic examination and weighed
- Since test substance-related organ weight changes were found in liver and spleen of highest dose group in F1 and F2 generations, these tissues were histopathologically examined for 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups
- If treatment-related histopathological changes were observed in the highest dose group, the same tissue in the next lower dose group was examined as well.
Statistics:
Bartlett's test: was applied for homogeneity of distribution for parametric data (body weight, food and water consumption, length of estrous cycle and gestation, precoital interval, the number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, behavioural test parameters, organ weight and sperm parameters);
For preweaning pups, body weight, AGD, viability, and age at the completion of developmental landmarks were similarly analyzed using the litter as the experimental unit.
One way analysis of variance was performed when the homogeneity of distribution was established.
If a significant difference was detected, Dunnett’s test was conducted for comparison between control and individual treatment groups.
Data without homogeneity were analyzed using the Kruskal-Wallis rank sum test. If significant differences were found, the Mann Whitneys’s U test was conducted for comparison between the control and each dose group.
Fisher exact test was used to compare the incidence of parental animals with clinical signs, and autopsy and histopathological findings, the incidence of females with normal estrous cycle, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ration and completion rate of negative geotaxis between the AS and control group.
The Wilcoxon rank sum test was used to analyze the incidence of pups with clinical signs and necroscopy findings per litter, the completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex.
Student's T-test was used to compare the number of primordial follicles in the control and highest dose group because the homogeneity of variance was indicated by the F-test.
All these statistical analysis were conducted using the 5% level of probability as the criterion for significance.
Reproductive indices:
- Copulation index (for males and females) (%): (no. of animals with successful copulation/no. of animals paired) x 100
- precoital interval (days)
- fertility index (for males and females) (%): (no. of males that impregnated a female or no. of pregnant/no. of animals with successful copulation) x 100
- Gestation index (%): (no. of females that delivered live pups/no. of pregnant females) x 100
- Gestation length (days)
- Delivery index (%): (no. of pups delivered/no. of implantations) x 100
- Estrous cycle in P0 and F1 females
Offspring viability indices:
For F1 and F2 offspring:
Maternal indices; no. of litters; no. of pups delivered; sex of all pups; sex ration of pups total (no. of male pups/total no. of pups)
Viability index calculated:
on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) x 100
on PND 4 (%) = (no. of live pups on PND 4/ no. of live pups on PND 0) x 100
on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no significant difference seen between control and AS treated groups in incidence of clinical signs; 120 ppm: one F1 female died (non adverse); 600 ppm: one P female died (non adverse); 3000 ppm: one F1 female died (non adverse)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: administered in drinking water
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant difference was seen between control and AS treated groups in the incidence of clinical signs of toxicity in either malel or female P and F1 rats.
120ppm: One F1 male died at 9 weeks of dosing. Soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. Necropsy revealed accumulation of ascitic and pleural fluid and dark purple discoloration of liver and kidneys
600ppm: One P female died at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this female from the beginning of 5 weeks of dosing
3000ppm: One F1 male died at 12 weeks of dosing. No clinical signs of toxicity were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
drinking water consumption: P males and females - significantly lower than control throughout the study (120-3000 ppm); F1 males - 120 ppm: significantly decreased during 3-6, 8, 10 weeks of dosing; 600, 3000 ppm: significantly decreased through dosing period; F1 females - 120 ppm: significantly decreased during 9-10 weeks of dosing; 600 ppm: significantly decreased during 10 week of dosing and 3 week of lactation; 3000 ppm: significantly decreased throughout the dosing period compared to controls
Food consumption: P males - 600ppm: significantly reduced during the first week of dosing; 3000ppm: significantly decreased during weeks 1,8 and 13-14; P females - 600ppm: significantly decreased during week 3 of lactation; 3000ppm: significantly decreased at 1 week of dosing and during week 3 of lactation; F1 males and females - 600 and 3000 ppm: significantly decreased in the 10 week of dosing (F1 males); significantly decreased in the 3 week of lactation (F1 females);
body weight: P males and females - 3000 ppm: significantly decreased in the first 2 or 3 weeks of dosing; 120 (F1 males), 600 (F1 males and F1 females) and 3000 ppm (F1 males and F1 females) - no significant differences in body weight compared to control; 120 ppm, F1 females - significantly increased body weights during 6-8 weeks of dosing

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
se table1 under "any other information on material and methody including tables"

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
P and F1 females - 120, 600 and 3000 ppm:
estrous cycle: no significant deviations in the estrous cycle of P and F1 females were observed during the premating period. However, a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
P males - 3000 ppm: absolute number of cauda epididymal sperm - reduced significantly (253.8 ±61.3 × 106/cauda versus 286.3 ±40.3 ×106/cauda in the control); however when expressed as the number per gram of tissues, there was no significant change.
F1 males - 3000 ppm: absolute number of cauda epididymal sperm - no change was found compared to the control animals. Number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm - no significant differences between control and AS-treated groups in either P or F1 adults (Note: no details were provided on the results of these examinations).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
P and F1 parental generation - copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations, number of litters or pups delivered (see table2)
- no significant differences were observed between the control and AS-treated groups in either P and F1 generation.
Copulation was not observed:
- in the P males: control (n=2), 120 ppm (n=2), 3000 ppm (n=2) and in the P females: in the control (n=1);
- in the F1 males: in the control group (n=1), 120 ppm (n=2) 600 ppm (n=1) and 3000 ppm (n=3) and in the F1 females: in the 120 ppm group (n=1), 3000 ppm (n=1).
After successful copulation, no pregnancy was observed:
- in P females: in the control (n=1), 120 ppm (n=2), 3000 ppm (n=1);
- in F1 females: in the control (n=2), 120 ppm (n=4), 600 ppm (n=2) and 3000 ppm (n=2).
No live pups delivered were found for pregnant rats from :
- P female in the 120 (n=1), 600(n=1) and 3000 ppm (n=1);
- F1 female in the 120 ppm group (n=1).
Comments: Overall, there were no treatment-related effects on reproduction parameters.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P males - 3000 ppm
absolute and relative liver weights
- were significantly decreased;
absolute spleen weight
- was significantly decreased;
- no significant change in relative weight.
F1 males - 3000 ppm
absolute weight of the adrenals
- was significantly decreased;
- no significant change in relative weight.
F1 males - 600 ppm
absolute weight of the testes
- was significantly decreased;
- no significant change in relative weight.
P and F1 females - 120, 600 and 3000 ppm
- no changes in absolute or relative weights of organs compared to the control (data not shown).
P and F1 females - 3000 ppm
number of primordial follicles in the ovary
- no difference between AS-treated and controls (data not shown).

GROSS PATHOLOGY (PARENTAL ANIMALS)
P and F1 generations - No dose-related gross lesions were found in F0 or F1 adults.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P and F1 males and females - 3000 ppm:
Histopathological examination of the reproductive organs revealed no compound-related alterations.


Dose descriptor:
LOAEL
Effect level:
31.2 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: Decreased body weight gain, decreased food consumption
Dose descriptor:
NOAEL
Effect level:
8.06 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Dose descriptor:
LOAEL
Effect level:
31.2 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: Decreased preweaning body weight gain in the F1 and F2 males and females; decreased absolute and relative liver and spleen weights in he F1 and F2 males and females; delayed vaginal opening in F1 females
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
NOAEL
Effect level:
8.06 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
2 F1 pups showed malformations (non adverse)
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: F1 males and females - body weights were significantly lower on PND21; F2 females - body weights were significantly lower than controls on PND21
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: vaginal opening was significantly delayed (F1)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: absolute and relative liver weights and absolute spleen weight, absolute weight of thymus, kidney, testes and epididymides decreased; absolute weight of uterus decreased at 600 ppm; relative brain weight decreased
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
F1 and F2 generation - 120, 600 and 3000 ppm:
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation.

CLINICAL SIGNS (OFFSPRING)
F1 generation - During the in-life check of delivered pups, one control F1 pup experienced trauma in the perianal region and tail and one F1 pup had hemimelia and oligodactyly in the 120ppm group, but there was no significant difference between the control and AS-treated groups.
F2 generation - No malformed F2 pups were found in any group.

BODY WEIGHT (OFFSPRING)
F1 generation - 3000 ppm group, F1 males and F1 females
- body weights of male and female pups were significantly lower on PND 21 compared to the control.
F2 generation - 3000 ppm, F2 females
- body weights were significantly lower than controls on PND 21.
3000 ppm, F2 males
- there were no significant differences in body weights between the control and AS-treated groups during the preweaning period.

SEXUAL MATURATION (OFFSPRING)
F1 males and females - 3000 ppm
- vaginal opening was significantly delayed (31.4±1.7 compared to 29.5±2.1 days in control). At 3000 ppm body weight at the time of vaginal opening was slightly higher than the control (119.0 ± 13.3 versus 109.6 ± 11.6 g) although not statistically significant.
- 120, 600 and 3000 ppm
- no significant differences between control and AS-treated groups were noted regarding age at preputial separation and no changes were found in body weights at the time of preputial completion.

ORGAN WEIGHTS (OFFSPRING) (see table3 and 4)
F1 generation - 3000 ppm, males and females
body weight - significantly lower at scheduled sacrifice compared to the control;
absolute and relative liver weights - significantly lower than the control;
absolute spleen weight - significantly decreased in both males and females and, a significant decrease in the relative weight was observed in males;
the absolute weight of the thymus - decreased in both sexes;
absolute weight of the kidney, testes and epididymides (males) - decreased compared to the control;
absolute weight of the uterus - decreased at 600 ppm compared to control;
relative brain weight - significantly increased in both sexes.
F2 generation - 3000 ppm, males
mean body weight at sacrifice - significantly lowered in both sexes;
absolute and relative weights of the thymus and spleen - significantly decreased in males;
absolute weight of the liver and epididymides - significantly decreased;
relative brain weight - significantly increased.
120 ppm, males
relative thymus weight - significantly decreased but no dose-response relationship.
3000 ppm, females
absolute and relative weights of the liver, the absolute weights of the spleen, ovary and uterus - significantly decreased;
relative brain weight - significantly increased.
600 ppm, females
the absolute brain weight - significantly decreased.

GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations:
F1 males and females, F2 males and females
- no treatment-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period (data not shown).

HISTOPATHOLOGY (OFFSPRING)
F1 males and females, F2 males and females
- no dose-related histopathological changes in the liver or spleen of male and female F1 and F2 weanlings. (fpr details see table 3+4)

OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT:
F1 males and females; F2 males - 120, 600 and 3000 ppm
- the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening were not significantly different between the control and AS-treated groups.
F1 males and females, F2 males and females - 120, 600 and 3000 ppm
- the AGD and AGD per cube root of the body weight ratio were not significantly different between control and AS-treated groups in male and female F1 and F2 pups (data not shown).
F2 females - 120, 600 and 3000 ppm
-completion rates of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks were not significantly different between AS-treated groups and controls.
600ppm - completion rates of pinna unfolding on PND 2 was significantly lower (17.0±35.4%, compared with 45.8±46.9 in controls), but no dose-response relation was observed.

NEUROMOTOR DEVELOPMENT
F1 males and females - 120, 600 and 3000 ppm
- no significant changes were observed in the development of reflexes (surface righting reflex on PND5, negative geotaxis reflex on PND8 and midair righting reflex on PND 18);
- no significant changes were observed in the response times of surface righting and negative geotaxis reflexes.
F2 males and females - 120, 600 and 3000 ppm
- surface righting reflex on PND 5 and negative geotaxis reflex on PND 8 were achieved in all male and female F2 pups in all groups;
- no significant changes were found in the response time (data not shown).
F2 females - 600 ppm
- the mid-air righting reflex on PND 18 was not achieved by 1 female in one of three trials; however, there was no significant difference in the mean success rate between the control and 600 ppm group (100±0.0% versus 98.4±7.3%).

BEHAVIOR PERFORMANCE
F1 males and females - 120, 600 and 3000 ppm
Spontaneous locomotor activity was not significantly different between control and AS- treated groups at 10-min intervals and for 60 min.
Learning and memory performance in T-maze test
Pre-test swimming trials in the straight channel
- no differences between male and female rats in each group compared to the controls;
- no significant changes in the elapsed time to traverse the straight channel;
- in males, no significant changes in the elapsed time and number of errors on days 2–4;
- in females, the elapsed time and the number of errors was significantly lowered at 600 ppm on day 2, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 between control and AS-treated groups (data not shown).
Dose descriptor:
LOAEL
Generation:
other: F1 and F2
Effect level:
31.2 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: Decreased preweaning body weight gain in the F1 and F2 males and females; decreased absolute and relative liver and spleen weights in he F1 and F2 males and females; delayed vaginal opening in F1 females
Dose descriptor:
NOAEL
Generation:
other: F1 and F2
Effect level:
8.06 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Reproductive effects observed:
not specified

Table 2: Reproductive performance of P0 and F1 parental animals

 AS (ppm)

 

 0

 120

 600

 3000

 P generation

 

 

 

 

 

 No. of rats (male/female)

 

 24/24

 24/24

 24/24

 24/24

 Copulation index (%)

 males

 91.7

 91.7

 100

 91.7

 

 females

 95.8

 100

 100

 100

 Precoital interval (days)

 

 3.2 ± 1.1

 3.2 ± 1.8

 2.9 ± 1.3

 2.8 ± 1.6

 Fertility index (%)

 males

 95.5

 90.9

 100

 95.5

 

 females

 95.7

 91.7

 100

 95.8

 Gestation index (%)

 

 100

 95.5

 95.7

 95.7

 Gestation length (days)

 

 22.4 ± 0.5

 22.5 ± 0.6

 22.1 ± 0.4

 22.3 ± 0.5

 Delivery index (%)

 

 94.3 ± 5.6

 88.6 ± 21.0

 90.7 ± 20.8

 92.0 ± 20.5

 F1 generation

 

 

 

 

 

 No. of rats (male/female)

 

 24/24

 23/24

 24/24

 24/24

 Copulation index (%)

 males

 95.8

 91.3

 95.8

 87.5

 

 females

 100

 95.8

 100

 95.8

 Precoital interval (days)

 

 3.3 ± 3.2

 3.0 ± 2.0

 2.7 ± 1.5

 2.3 ± 1.1

 Fertility index (%)

 males

 91.3

 81.0

 91.3

 95.2

 

 females

 91.7

 82.6

 91.7

 91.3

 Gestation index (%)

 

 100

 94.7

 100

 100

 Gestation length (days)

 

 22.4 ± 0.5

 22.3 ± 0.5

 22.2 ± 0.4

 22.2 ± 0.4

 Delivery index (%)

 

 94.0 ± 9.9

 87.5 ± 22.6

 91.4 ± 10.7

 94.6 ± 6.8

Table 3: Absolute and relative organ weight of F1 and F2 male weanlings (% of control)

 As (ppm)

 0   

 120   

 600   

 3000   

 Organ weight

 F1 males

 F2 males

 F1 males

 F2 males

 F1 males

F2 males

 F1 males

 F2 males

 number of animals

 22

 21

 20

 18

 22

 22

 22

 21

 body weight (g)

 100%

 100%

 NS

 NS

 NS

 

 87.44**

 90.31**

 brain                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

relative weight (g/100g bw) 

 100%

 100%

 NS

 NS

 NS

 NS

 113.22**

 112.11**

 thymus                        

 absolute weight (g)

 100%

 100%

 NS

 

 NS

 NS

 81.33**

 79.84**

 relative weight (g/100g bw)

 100%

 100%

 NS

 89.29*

 NS

 NS

 NS

 87.92**

 Livera                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 80.60**

 87.78**

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 91.61**

 NS

 Kidneya                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 89.62**

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 spleen                         

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 76.40**

 80 .43

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 86.93**

 88.36**

 testisa                        

absolute weight (g) 

 100%

 100%

 NS

 NS

 NS

 NS

 90.44*

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 Epididymisa                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 88.02**

 93.62*

relative weight (g/100g bw) 

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

NS- not statistically significant compared to untreated control

**- significantly different from control, p<0.05

*- significantly different from control, p< 0.01

Table 4: Absolute and relative organ weight of F1 and F2 female weanlings (% of control)

 As (ppm)

 0   

 120   

 600   

 3000   

 Organ weight

 F1 females

 F2 females

 F1 females

 F2 females

 F1 females

F2 females

 F1 females

 F2 females

 number of animals

 22

 22

 20

 18

 22

 21

 21

 21

 body weight (g)

 100%

 100%

 NS

 

 NS

 

 89.91**

 91.34**

 brain                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 102.5*

 NS

 NS

relative weight (g/100g bw) 

 100%

 100%

 NS

 NS

 NS

 

 110.20**

 110.05**

 thymus                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 81.72**

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 Livera                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 84.80**

 86.24**

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 94.26*

 94.56**

 Kidneya                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 spleen                         

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 86.65**

 84.11

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

ovarya                      

absolute weight (g) 

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 84.52**

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

                       uterusa 

 absolute weight (g)

 100%

 100%

NS

NS 

 83.85*

 NS

 78.47**

 81.49*

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

NS- not statistically significant compared to untreated control

**- significantly different from control, p<0.05

*- significantly different from control, p< 0.01

Conclusions:
Aluminium sulfate had no effect on reproductive performance
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the fact that following absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining, factor the systemic effects of aluminium, including acute toxicity common (refer to endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in the Annex VIII-IX, in accordance with Annex XI, 1.5 of Regulation (EC) No. 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

As no studies investigating toxicity to reproduction of reaction mass of aluminium htdroxide and aluminium nitrate and aluminiumm sulphate are available in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from supporting substances (structural analogues) e.g. aluminium compounds was considered. Aluminium oxide, aluminium hydroxide and aluminium metal are insoluble in water under standard conditions. Based on these physico-chemical characteristics, it is likely that under physiological conditions, the absorption and associated bioavailability of aluminium hydroxide, aluminium oxide and aluminium metal will be low. Following oral absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining factor the systemic effects of aluminium, including acute toxicity. Hence, it can be assumed that Al3+is the substance of biological interest and the toxicological effects can be attributed mainly to Al3+.

Following absorption of the substance used for read-across like aluminium salts (e.g., aluminium nitrate, aluminium chloride, aluminium sulphate, etc.) aluminium is present in the body as Al3+as well. Therefore, with appropriate consideration of bioavailability differences, it is reasonable to consider data obtained from aluminium salts, generally more soluble, in the hazard identification of the highly soluble aluminiumsulfatenitrate.

In conclusion, in terms of hazard assessment of toxic effects, available data for human health endpoints for various aluminium compounds can be read-across to reaction mass of aluminium nitrate and aluminium sulphate since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+) (Krewski et al., 2007; ATSDR, 2008).

A detailed justification read-across is provided in the technical dossier (see IUCLID Section 13) as well as in the Chemical Safety Report (see Part B).

 

Since no studies investigating the toxicity to reproduction of reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate are available in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from supporting substance (structural analogue), aluminium sulphate (10043-01-3) was performed. Read-across is justified based on the fact that following oral absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining factor the systemic effects of aluminium, including toxicity to reproduction.

 

10043-01-3 (aluminium sulphate)

In a two-generation reproductive toxicity study conducted with a method similar to OECD guideline 416 and Japanese guideline for the “designation of food additive and for the revision of standards for the use of the food additives” and GLP conform (Hirata-Koizumi, 2011), male and female rats were given aluminium sulfate in drinking water at 0, 120, 600 or 3000 ppm. The administration of test substance has as effect the reduction of the water consumption in all treatment groups, and the transient decreased of the body weight in in 3000 ppm, while the liver and the spleen weights were decreased at weaning. At this dose, vaginal opening resulted slightly delayed. No other compound-related effects were observed in other reproductive parameters. The data indicated that the NOAEL of the test substances is 600 ppm for parental systemic toxicity and reproductive toxicity. The total ingested dose of aluminium from drinking water and food (standard rat diet, containing 25-29 ppm of aluminium), combined for this 600 ppm groups was calculated to be 8.06 mg Al/kg bw/day.

 

Additionally, data from structurally related substances were used as supporting information.

 

 

CAS 31142-56-0 (aluminium citrate)

No effects on the length of the gestation period in female rats exposed to up to 3225 mg/kg bw/day of aluminium citrate (equivalent to 300 mg Al/kg bw/day) were reported in the combined developmental neurotoxicity/chronic study similar to OECD guidelines 426 and 452 and in compliance with GLP conducted with aluminium citrate as described in the "Developmental toxicity" section below (ToxTest Alberta Research Council, 2009).

CAS 1327-41-9 (aluminium chloride, basic)

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD guideline 422, GLP compliant) was conducted in rats exposed to aluminium chloride basic via oral gavage (Beekhuijzen, 2007).

Four groups of 20 Wistar rats (10 per sex) were exposed to aluminium chloride basic at 0, 40, 200 and 1000 mg/kg bw/day, corresponding to 0, 3.6, 18 and 90 mg Al/kg bw/day. Males were exposed for 28 days (starting 2 weeks prior to mating, during mating and up to termination). Females were exposed for 37-53 days (starting 2 weeks prior to mating, during mating, during post-coitum and during at least 3 days of lactation).

Histopathological assessment revealed a mild to moderate subacute inflammation of the glandular stomach mucosa and minimal to moderate superficial mucosal eosinophilic spheroids in both sexes at 90 mg Al/kg bw/day. In males, these findings supported the macroscopic findings of the glandular mucosa/limiting ridge at this dose level. The mucosal eosinophilic spheroids are apparently intracellular degenerative products of the superficial mucosa and possibly associated with inflammation below the base of the mucosa. These findings are indicative of local irritating properties of aluminium chloride basic.

The slightly lower body weight and food intake of females at 90 mg Al/kg bw/day recovered to control levels as treatment progressed. No toxicological significance was therefore ascribed to these changes.

Changes in clinical pathology parameters at 90 mg Al/kg bw/day were of a slight nature and generally within the range expected for rats of this age and strain. Also, any morphological correlates were absent. Therefore, these changes were considered not indicative of organ dysfunction and to be of no toxicological significance.

There were no (further) changes for mortality, clinical signs, functional observations, organ weights, reproduction breeding data and pup development that were considered to be an effect of treatment.

In conclusion, treatment with aluminium chloride basic by oral gavage in male and female Wistar rats at dose levels of 40, 200 and 1000 mg/kg bw/day, corresponding to 3.6, 18 and 90 mg Al/kg bw/day, revealed parental toxicity at the highest dose level comprising local stomach effects. No reproduction, breeding and developmental toxicity was observed for treatment up to the highest dose level.

Based on the findings on the stomach observed macroscopically, the parental NOAEL for local effects was established at 18 mg Al/kg bw/day. The parental NOAEL for systemic toxicity was 90 mg Al/kg bw/day. The reproduction, breeding and developmental NOAEL was established at 90 mg Al/kg bw/day, the highest dose tested in this study.

 

CAS 7784-27-2 (aluminium nitrate nonahydrate)

Aluminium nitrate was tested for its effects on reproduction, gestation and lactation in rats, at the following doses: 0, 180, 360 and 720 mg/kg bw/day (Domingo, 1987). Adult male rats were treated orally by gavage for 60 days prior mating with mature virgin females rats treated for 14 days treated for prior to mating with treatment continuing throughout mating, gestation, parturition, and weaning of the litters. One-half of the dams in each group were killed on day 13 of gestation and the remaining dams were allowed to deliver and wean their offspring. Postnatal development was monitored. No adverse effects on fertility or general reproductive parameters were evident at doses used in this study.

The survival ratios were higher for the control group. Moreover, a dose-dependent delay in the growth of the living young could be observed in aluminium treated groups. However the effects were negligible and transient(slight decreases in body weight, body length, and tail length observed on postpartum days 1 and 4 were no longer evident at time of weaning).

In another study the reproductive toxicology of aluminium was studied in mice (Llobet, 1995). Adult male mice were treated intraperitoneally with test substance at doses of 0, 50 100, and 200 mg/kg/day during 4 weeks before mating with untreated females. Decreased body weight was observed in all treated groups. Decreased pregnancy rate was notice in females mated with males previously mated with 100 or 200 mg/kg/day of aluminium nitrate. Male mice treated with high dose of test material revealed significantly loss in testicular and spermatid counts and epididymal sperm counts. Spermatid counts were also decreased at 100 mg/kg/day.

Nevertheless, the sperm motility was unaffected, and the percentages of the morphological normal spermatozoa in all mice exposed to test substance were comparable to those found in control mice.

Histological changes, including necrosis of spermatocytes/spermatids, were observed in the testes of male mice treated with high dose of aluminium nitrate (100 and 200 mg/kg/day), whereas the tubular diameters were unaffected by test substance administration. The NOAEL was 50 mg/kg/day.

Short description of key information:

OECD 416, rat: NOAEL: 8.06 mg/kg bw/day (test material: Al) of the read across substance aluminium sulfate

Justification for selection of Effect on fertility via oral route:

Hazard assessment is conducted by means of read across from a structural analogue aluminium sulfate (CAS 10043-01-3). The available study is adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details)  

Effects on developmental toxicity

Description of key information

Combined OECD 426 and OECD 452 , rat: NOAEL:  3225 mg/kg bw/day corresponding to 300 mg/kg bw/day (test material: Al) of the read across substance aluminium citrate.

Link to relevant study records
Reference
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: 242.5 - 333.4 g (females, target 160-360 g); 335.4 - 470.8 g (males; target 245-585 g).
- 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.
- 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
- Humidity (%): 30 - 70
- Air changes (per hr): ≥ 10 per hour in the room and 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): 12/12

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.

The stability and homogeneity of the dosing solutions under test conditions were determined in a separate study. 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)
All analyses were appropriately blinded.
Details on mating procedure:
- Impregnation procedure: cohoused. Sires and dams were allocated into breeding pairs by using SAS PROC PLAN procedure.
- If cohoused:
- Length of cohabitation: animals were allowed to breed for up to five consecutive nights.
- Proof of pregnancy: vaginal plug. The date of breeding (i.e. insemination) was defined as the day when a vaginal plug was first detected.
-other: 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). Upon detection of a plug, breeding pairs were separated.

Duration of treatment / exposure:
Dams
Day 6 of gestation – day 21 post-natal

Pups (males and females)
Day 22 – 364 post-natal
Cohort 1: Day 6 of gestation – day 21 post-natal, day 1-22 post-natal
Cohort 2 : Day 6 of gestation – day 21 post-natal, day 1-64 post-natal
Cohort 3: Day 6 of gestation – day 21 post-natal, day 1-120 post-natal
Cohort 4 : Day 6 of gestation – day 21 post-natal, day 1-364 post-natal
Frequency of treatment:
daily, 7 days per week
Duration of test:
1 year
Remarks:
Doses / Concentrations:
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
Basis:
nominal in water
No. of animals per sex per dose:
20 P females;
10-20 F1 females and 10-20 males
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 based on the results of a foregoing 90 day 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.
Statistics:
See "any other information on materials and methods incl. tables"
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: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
3 225 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
322.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
LOAEL
Effect level:
1 075 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined
Dose descriptor:
other: Embryotoxic / teratogenic effects:not examined
Based on:
other: Embryotoxic / teratogenic effects:not examined
Basis for effect level:
other: Embryotoxic / teratogenic effects:not examined
Remarks on result:
not measured/tested
Abnormalities:
not specified
Developmental effects observed:
not specified

Offspring results:

Mortality

Mortalities/unscheduled euthanizations observed in each group.

 

Female

Male

 

Died

Euthanized

Died

Euthanized

Control

4

4

3

1

Low-Dose

1

1

2

3

Mid-Dose

0

0

2

0

High-Dose

4

9

8

37

Na-citrate

3

2

7

3

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

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

Clinical Observations

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

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

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

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

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

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

Body Weight

Pre-weaning phase:

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

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

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

Post-weaning phase:

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

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

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

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

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

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

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

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

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

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

Growth Curve Parameters

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

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

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

Water Consumption

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

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

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

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

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

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

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

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

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

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

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

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

Actual Doses Received

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

Males

Group

Week 1

Week 7

Week 14

Week 28

Week 49

Low-Dose

134%

57%

37%

20%

17%

Mid-Dose

174%

84%

51%

28%

23%

High-Dose

165%

117%

-

-

-

Females

Low-Dose

145%

60%

57%

34%

33%

Mid-Dose

199%

74%

64%

38%

41%

High-Dose

205%

118%

93%

58%

42%

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

Organ Weight

Brain weights.

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

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

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

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

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

Pathomorphology and Histology

Necropsy Results

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

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

Females

Day 23

Day 64

Day 120

Day 364

Control

0

1

0

0

Low-Dose

0

0

0

0

Mid-Dose

0

1

0

0

High-Dose

0

3

2

3

Na-citrate

0

0

1

0

Males

Day 23

Day 64

Day 120

Day 364

Control

0

0

0

0

Low-Dose

0

0

0

1

Mid-Dose

0

3

1

0

High-Dose

0

11

7

5

Na-citrate

0

1

0

0

Urinary tract pathology was a treatment-related effect.

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

Histopathological examination of CNS tissue and muscle (microscopic)

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

Day 64 cohort:

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

Low dose group - All tissues were normal.

Mid-dose group - All tissues were normal.

High dose group - All tissues were normal.

Na-citrate group - All tissues were normal.

Day 120 cohort:

Control group – All tissues normal.

Low-dose group - All tissues were normal.

Mid-dose group - All tissues were normal.

High-dose group - All tissues were normal.

Na-citrate group - All tissues were normal.

Day 364 cohort:

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

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

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

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

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

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

Group

Sex

Day 364

Control

M

2

 

F

3

Low-Dose

M

2

 

F

4

Mid-Dose

M

1

 

F

3

High-Dose

M

n/a

 

F

3

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

Developmental Landmarks

Females

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

Males

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

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

FOB (neonatal pups)

Females

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

Males

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

FOB (juveniles)

Day 364 cohort

Females

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

Males

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

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

FOB (adult pups)

Day 364 cohort

Females

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

FOB Parameter Group effect Pairwise Differences

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

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

- treatment-related effect

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

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

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

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

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

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

Defecation characteristics P<0.0001 As above

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

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

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

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

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

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

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

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

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

Males

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

FOB Parameter Group effect Pairwise Differences

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

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

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

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

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

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

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

Defecation characteristics P<0.0001 Not clearly related to treatment.

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

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

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

Urine characteristics P<0.0001 Not clearly related to treatment.

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

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

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

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

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

Motor Activity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Auditory Startle Response

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

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

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

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

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

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

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

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

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

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

T-maze

The T-maze testing was conducted at PND 21.

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

Group

Male

Female

Control

42.11

26.32

Low-Dose

25.00

42.11

Mid-Dose

31.58

47.37

High-Dose

63.16

31.25

Na-citrate

26.32

50.00

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

Morris Water Maze

Training Trial Latencies

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

Platform-Removed Probe Test Search Strategies

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

Platform Visible Latencies

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

Platform Visible Type of Search

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

 

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

Haematology

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

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

Day 23 cohort:

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

Day 64 cohort:

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

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

the low-dose and the mid-dose groups.

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

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

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

The anaemia had resolved in the females.

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

Coagulation parameters:

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

Conclusions:
The results from this study are informative for developmental and neurotoxic effects due to prenatal and chronic postnatal exposure of rats to high doses of aluminium citrate 3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day). 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. A LOAEL of 1075 mg AlCitrate/kg bw/day (100 mg Al/kg bw/day) for aluminium toxicity is assigned based on this study. 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 (1075 mg AlCitrate/kg bw/day; 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).

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. As such, 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.

No treatment-related differences in FOB characteristics were observed in the neonatal and juvenile pups.
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 of aluminium citrate 3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day). Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al citrate and Na-citrate were administered to Dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.

 

Endpoints and observations in the dams included water consumption, body weight, a Functional Observational Battery (FOB), morbidity and mortality. Endpoints were assessed in both female and male pups that targeted behavioural 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 tests in the dams. Due to the differences in water consumption, the % of target dose differed between groups and with time through the study. In the high dose group of dams, the actual dose during the first week of gestation was 200 mg Al/kg bw/day, 67% of the target dose (300 mg Al/kg bw/day). In the last week before weaning (and sacrifice), the actual dose received by the dams was close to 175% of the target dose. Statistical analyses comparing the actual doses received by the low, mid- and high- Al-citrate treatment groups showed that the order of the dose groups was maintained, however.

 

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

 

In the female pups, the mean number of days to reach vaginal opening was 31.3 (±2.1, sd) in the control group and 39.7 (±5.6, sd) in the high dose Al-citrate group, a significant difference (p<0.0001). In males, the mean number of days to reach preputial separation was 39.6 (±2.1, sd) in the control group and 42.5 (±3.2, sd) in the high dose group, also a significant difference in the pair-wise comparisons (p<0.0001). Delayed development of both male and female pups wasobserved in the high dose Al-citrate group and also in the Na-citrate group. The effect is considered treatment-related but whether the effect is secondary to decreases in body weight is not clear, however. In addition, as an effect was observed in the Na-citrate group, the role of aluminium in causing this effect can neither be concluded nor excluded.

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 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. 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.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. Overall, these deviations were unlikely to have impacted the results of the study.

 

The results from this study are informative for neurotoxic effects due to combined prenatal and chronic postnatal exposure of rats to high doses of aluminium citrate 3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day).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 1075 mg AlCitrate/kg bw/day (100 mg Al/kg bw/day) for aluminium toxicity is assigned. The critical effect was a deficit in fore- and hind-limb grip strength in the mid-dose group, supported by evidence of dose response and less consistently observed effects in the mid-dose animals: urinary tract lesions at necropsy (4 males, 1 female); body weight (mid-dose males weighed less than controls in the Day 120 cohort); defecation (more boluses produced by females in the mid-dose group compared with the controls); urination (mid-dose males produced more urine pools than controls); tail pinch (mid-dose females displayed more exaggerated responses); foot-splay (mid-dose females had significantly narrower foot-splay than the controls); and the albumin/globulin ratio (Day 64 mid-dose males had a greater mean ratio than the controls).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the fact that following absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining, factor the systemic effects of aluminium, including acute toxicity common (refer to endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in the Annex VIII-IX, in accordance with Annex XI, 1.5 of Regulation (EC) No. 1907/2006.
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

As no studies investigating developmental toxicity/teratology of reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate are available in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from supporting substances (structural analogues) e.g. aluminium compounds was considered. Aluminium oxide, aluminium hydroxide and aluminium metal are insoluble in water under standard conditions. Based on these physico-chemical characteristics, it is likely that under physiological conditions, the absorption and associated bioavailability of aluminium hydroxide, aluminium oxide and aluminium metal will be low. Following oral absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining factor the systemic effects of aluminium, including acute toxicity. Hence, it can be assumed that Al3+is the substance of biological interest and the toxicological effects can be attributed mainly to Al3+.

Following absorption of the substance used for read-across like aluminium salts (e.g., aluminium nitrate, aluminium chloride, aluminium sulphate, etc.) aluminium is present in the body as Al3+as well. Therefore, with appropriate consideration of bioavailability differences, it is reasonable to consider data obtained from aluminium salts, generally more soluble, in the hazard identification of the highly soluble aluminiumsulfatenitrate.

In conclusion, in terms of hazard assessment of toxic effects, available data for human health endpoints for various aluminium compounds can be read-across to reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+) (Krewski et al., 2007; ATSDR, 2008).

A detailed justification read-across is provided in the technical dossier (see IUCLID Section 13) as well as in the Chemical Safety Report (see Part B).

 

Since no studies investigating the investigating developmental toxicity/teratology of reaction mass of aluminium nitrate and aluminium sulphate are available in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read-across from supporting substance (structural analogue), aluminium sulphate (10043-01-3) was performed. Read-across is justified based on the fact that following oral absorption, aluminium is present in the body as the ionic species (Al3+), which is the determining factor the systemic effects of aluminium, including developmental toxicity/teratology.

 

CAS 31142-56-0 (aluminium citrate)

A developmental neurotoxicity and chronic toxicity study similar to OECD guidelines 426 and 452 and in compliance with GLP was conducted with aluminium citrate (ToxTest. Alberta Research Council Inc., 2009). Pregnant Sprague-Dawley dams were administered aqueous solutions of aluminium citrate at 3 dosage levels (nominal - 322.5, 1075 and 3225 mg/kg bw/day, equivalent to 30, 100 and 300 mg Al/kg bw/day, respectively). Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L) or plain water (control group). The Al-citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Pups were assigned to one of four cohorts (80 males, 80 females): a pre-weaning cohort that was sacrificed at PND 23, and cohorts that were sacrificed at PND 64, PND120 and PND 364.

Endpoints and observations in the dams included water consumption, body weight, a Functional Observational Battery (FOB), morbidity and mortality. Endpoints were assessed in both female and male pups that targeted behavioural 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 (expressed as equivalent aluminium 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 F1 adult males. Higher mortality and significant morbidity were observed in the F1 adult males 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 compared to controls. 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 females of the F1 generation. In the Day 120 cohort males, the mid-dose animals were significantly lighter than the controls. An effect of Na-citrate was observed in the female of the F1 generation 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), but a similar difference was seen for the Na-citrate control group. 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); a similar delay was also observed in the Na-citrate control group. Delayed development of both male and female pups was observed in the high dose Al-citrate group and also in the Na-citrate group.Additionally, a litter effect was noted by the authors. The effect is considered treatment-related but whether the effect is secondary to decreases in body weight is not clear, however. In addition, as an effect was observed in the Na-citrate group, the role of aluminium in causing this developmental effect can neither be concluded nor excluded.

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 at PND 22. In the adult F1 animals, 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. However, a similar effect was observed in the Na-citrate controls as well. 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. Furthermore, the FOB endpoint most consistently associated with Al-citrate treatment, grip strength, measurements showed considerably variability and a consistent ordering of the Al-treatment group responses (dose-response) was not observed at all time points. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning Day 23 cohort) or the Morris Water Maze test (Day 120 cohort).

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

Whole body Al levels in neonatal pups from high dose females and males were greater than those in the control groups. There were no significant sex differences. These results suggest transfer of Al from dams to pups in utero, although a contribution from breast milk PND 0 to 4 is also possible. 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.

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. Overall, these deviations were unlikely to have impacted the results of the study.

The results from this study are informative for neurotoxic effects due to combined prenatal and chronic postnatal exposure of rats to high doses of aluminium (30 mg Al/kg bw/day, 100 mg Al/kg bw/day and 300 mg Al/kg bw/day). As the offspring were dosed during the whole post-weaning period, it is difficult to differentiate between developmental or direct toxicity after weaning, however. Urinary tract pathology was observed in rats in the high dose group, more frequently and more severe in the males. The study showed no evidence of an effect of Al-citrate on memory or learning but a more consistent effect was observed in endpoints in the neuromuscular domain.

The ambiguity as to the critical period of exposure and the time-varying water consumption complicate the derivation of a point-of-departure from this study. A LOAEL for kidney toxicity and possible neurotoxicity (based on the effects on grip strength) of 1075 mg/kg bw/day of aluminium citrate (equivalent to 100 mg Al/kg bw/day) for aluminium toxicity was assigned. The critical neurological effect was a deficit in fore- and hind-limb grip strength in the mid-dose group, supported by evidence of dose response – although the results were variable and may be related to body weight effects as well – and 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).

Consequently, the NOAEL for neurotoxicity was 322.5 mg/kg bw/day of aluminium citrate (equivalent to 30 mg Al/kg bw/day). Furthermore, based on the lack systemic effects in general and of effects on gestational length in particular and the absence of clinical signs in maternal animals, 3225 mg/kg bw/day of aluminium citrate (equivalent to 300 mg Al/kg bw/day) was considered the maternal NOAEL for reproduction toxicity.

As no effects were observed in the F1 generation up to the juvenile age, the effects observed in this study appear to be related to repeated direct substance intake of the F1 generation rather than to intra-uterine or lactational exposure.

 

Additionally, data from structurally related substances were used as supporting information.

 

10043-01-3 (aluminium sulphate)

In a two-generation reproductive toxicity study conducted with a method similar to OECD guideline 416 and Japanese guideline for the “designation of food additive and for the revision of standards for the use of the food additives” and GLP conform (Hirata-Koizumi, 2011), male and female rats were given aluminium sulfate in drinking water at 0, 120, 600 or 3000 ppm. The administration of test substance has as effect the reduction of the water consumption in all treatment groups, and the transient decreased of the body weight in in 3000 ppm, while the liver and the spleen weights were decreased at weaning. At this dose, vaginal opening resulted slightly delayed. No other compound-related effects were observed in other reproductive parameters. There were no compound-related changes in developmental parameters. The data indicated that the NOAEL of the test substances is 600 ppm for developmental toxicity. The total ingested dose of aluminium from drinking water and food (standard rat diet, containing 25-29 ppm of aluminium), combined for this 600 ppm groups was calculated to be 8.06 mg Al/kg bw/day.

 

CAS 7784-27-2 (aluminium nitrate nonahydrate)

The effects of oral aluminium ingestion on perinatal and postnatal development were evaluated in a reliable and well conducted study which meets basic scientific principles (Domingo, 1987). Aluminium nitrate was administered by gavage to four groups of pregnant Sprague-Dawley rats from the day 14 of gestation through 21 days of lactation at doses of 0, 180, 360 and 720 mg/kg/day. The doses used did not produce overt fetotoxic effects. However, the growth of the offspring was significantly less from birth and during all the period of lactation for the higher doses of aluminum nitrate. Very few toxic effects could be observed for the 180 mg/kg/day group. On the basis of the above exposed the NOEAL was calculated to be 180 mg/kg bw/day corresponding to13 mg/kg bw/day Al.

In another study conducted to determine the embriotoxic and teratogenic potential of aluminium, pregnant rats were treated with by gavage with a daily dose of 180, 360, or 720 mg/kg bw of aluminium nitrate from day 6-14 of gestation (Paternain, 1988). Fetal examinations were conducted on day 20 of gestation. The number of corpora lutea, total implants, and resorptions as well as the number of live and dead fetuses in the treated animals resulted to be not significantly different from the control group. Therefore, embryo lethality of aluminium cannot be induced (as measure of percent dead and resorbed fetuses). However, exposure of rats to aluminium nitrate resulted in decreased fetal body weight and increased the incidence and types of external, visceral, and skeletal malformation and variations in all the treated groups. As a decrease in maternal weight gain was also reported at all dose level the teratogenic effects of aluminium nitrate administration in rats given doses were induced in concomitance with maternal toxicity. The LOAELs for embryotoxicity and teratogenicity were 180 and 360 mg/kg bw/day respectively corresponding to 13 and 26 mg/kg bw/day Al.

 

CAS 21645-51-2 (aluminium hydroxide)

Aluminium hydroxide was administered by gavage (2 times, daily) to pregnant Sprague-Dawley rats at dose levels of 192 (n = 18 animals per group), 384 (n = 18 animals per group) and 768 (n = 10 animals per group) mg/kg (equivalent to 66.5, 133 and 266 mg Al/kg bw/day, respectively) from day 6 - 15 of gestation in an acceptable, well conducted study which meets basic scientific principles (Gomez, 1990). The animals were killed on day 20 of gestation. No adverse effects were reported on animal appearance, behaviour, maternal body weight, or absolute and relative organ weight (uterine, kidney and liver). No differences were observed for haematological and biochemical parameters but detailed results for these outcomes were not provided in the publication. Although not statistically significant, the incidence of early resorptions was higher in all Al(OH)3-treated groups than in the control group (0.4 - in the 46 mg Al/kg group, 1.3 - in the 92 mg Al/kg group, and 0.6 – in the 266 mg Al/kg group versus 0.0 in the control group). Increased post-implantation loss (%) was observed compared to the control group (3.6 - in the 46 mg Al/kg group, 12.5 - in the 92 mg Al/kg group, and 5.0 – in the 266 mg Al/kg group versus 0.6 in the control group). Observed changes were not considered as treatment related effects because no relationship to dose was observed. Increased post-implantation loss (2.2 times compared to the control group) was observed only in the dose 92 mg Al/kg group. Statistically significant decrease in maternal food consumption was not associated with decreased maternal body weight and no dose-response relationship was found. No Al-treatment related effects were observed on critical gestational parameters such as number of litters, corpora lutea, number of total implantations, number of live fetuses, sex ratio, or fetal body weight at any dose administered. During fetal examination, no external and visceral abnormalities or skeletal malformation was detected. No significant differences in placental concentrations of aluminium were observed between the different groups. The NOAEL was considered to be 266 mg Al/kg bw/day based on lack of embryo/fetal toxicity or teratogenicity.

The influence of citric acid on the embryonic and/or teratogenic effects of high doses of Al(OH)3 in rats was investigated by Gómez et al. (1991). Three groups of pregnant rats were administered daily doses (gavage) of Al(OH)3 (384 mg/kg bw/day, equal to 133 mg Al/kg bw/day , n = 18), aluminium citrate (1064 mg/kg bw/day, n = 15), or Al(OH)3 (384 mg/kg bw/day, equal to 133 mg Al/kg bw/day) concurrently with citric acid (62 mg/kg bw, n = 18) on gestational days 6 to 15. A control group received distilled water during the same period (n = 17). There were no treatment-related differences on critical gestational parameters such as numbers of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, or fetal body weight in the group treated with Al(OH)3. No external and visceral abnormalities or skeletal malformation were detected on fetal examination. Maternal and fetal body weights were significantly reduced, the number of fetuses with delayed sternabrae and occipital ossification was significantly increased (p < 0.05), the number of fetuses with absence of xiphoides was increased in the group treated with Al(OH)3 and citric acid as compared to the control group. No significant differences in the number of malformations were detected between any of the groups (authors did not provide the quantitative data). 

 

 

CAS 21645-51-2 (aluminium hydroxide) and CAS 18917-91-4 (aluminium lactate)

In another well conducted study which meets basic scientific principles the developmental toxicity of high doses of aluminium was evaluated (Colomina, 1992). In this study the influence of lactate on developmental toxicity attributed to high doses of Al(OH)3 in mice. Oral (gavage) daily doses of Al(OH)3 (166 mg/kg bw, n = 11), aluminium lactate (627 mg/kg b, n = 10), or Al(OH)3 (166 mg/kg bw) with lactic acid (570 mg/ kg bw, n = 13) were administered to pregnant mice from gestational day 6 to 15 was evaluated. An additional group of mice received lactic acid alone (570 mg/kg bw). A control group (n = 13) received distilled water during the same period. No signs of maternal toxicity (no statistically significant changes in food consumption, maternal body and organ weight) were observed in the dams treated with Al(OH)3. No statistically significant treatment-related differences on critical gestational parameters such as number of litters, corpora lutea, number of total implantations, number of live fetuses, sex ratio, or fetal body weight were observed in the Al (OH)3-treated group and no external abnormalities or skeletal malformation were detected on fetal examination. However, aluminium concentrations were significantly higher in the bones of dams, and aluminium was detected in the whole fetus of the Al(OH)3-treated animals. Concurrent administration of Al(OH)3 and lactic acid resulted in significant reductions in maternal weight compared to the control group. In the group given lactate only, aluminium was detected in whole fetuses; however, this was not statistically different from the mean level found in the control group. Aluminium lactate administration resulted in significant decreases in maternal body weight and food consumption, fetal body weight accompanied by increases in the incidence of cleft palate. Delayed ossification was also observed in the aluminium lactate-treated animals. Although not statistically significant, the incidence of skeletal variations was higher in the group concurrently administered Al(OH)3 and lactic acid than in the control group. No other signs of developmental toxicity were detected in the Al (OH)3 and lactic acid group.

Justification for selection of Effect on developmental toxicity: via oral route:

Hazard assessment is conducted by means of read across from a structural analogue aluminium citrate (31142-56-0). The available study is adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details)  

Justification for classification or non-classification

The available data on toxicity to reproduction of substances structurally realted to reaction mass of aluminium hydroxide and aluminium nitrate and aluminium sulphate do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and therefore are conclusive but not sufficient for classification.

Additional information