Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Strengths of this study include the fact that it was conducted according to OECD TG 416 in accordance with GLP procedures, multiple dose levels were studied, both sexes were used, administered doses were analytically verified, a dose finding study was conducted and used doses were justified, stability of AAS in drinking solutions was detected and controlled. Limitations of this guideline study: urinalysis was not performed, lack of examinations/evaluation data on clinical signs of toxicity and absence of pH values for the AAS-containing drinking solution detract from the report. Few details were provided on the statistical methods used, blinding of observations, selection of the animals for the neurobehavioral tests, sperm parameters examination and the fact no measures of Al levels were conducted in the body fluids/organs. Possible irritant effects of AAS on the gastrointestinal tract mucosa of rats based on the results from the preliminary range-finding study could not be excluded. 

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
urinanalysis was not performed
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Aluminium Ammonium sulfate (AAS)
- Analytical purity: 99.5 %
- Lot/batch No.: A81009
- Stability under test conditions: AAS was stable in deionized water at concentrations of 0.05 and 10 mg/mL at least 12 days (5 days storage at room temperature following 7 day refrigerated storage)
- Storage condition of test material: the test material was kept in a sealed container under cool and dark conditions

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles river Laboratories Japan, Inc.
- Age at study initiation: (P) 5 wks
- Housing: animals were housed individually, except for the acclimation, mating and nursing periods, in metal-bracket-type cages with wire-mesh floor
- Diet: standard rat diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum, Al content in diet, analyzed by flame atomic absorption spectrometry for each lot of diet, ranged from 22 ppm to 29 ppm
- Water: deonized drinking water with (treatment group) or without (control group) dose concentrations, water given to controls contained < 5 µg Al/mL
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: fresh dosing solutions were prepared at least once every 7 days and the drinking water was replaced at least once every 5 days
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until successful copulation occured or the mating period of 2 weeks had elapsed
- Proof of pregnancy: vaginal smears were examined daily for presence of sperm, and the presence of sperm in the vaginal smear and/or a vaginal plug were considered as evidence of successful mating, detection of sperm in the vaginal lavage was designated as day 0 of gestation
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged: from day 17 of gestation to day 21 after delivery, dams and litters were reared using wood chips as bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
concentrations of AAS in drinking water were analyzed in the first and last preparations and once every 3 months, ans verified using high performance liquid chromatography (the quantitation limit -5 µg/mL)
Duration of treatment / exposure:
F0 males: 10 weeks prior to mating, during mating and up to termination after the parturition of paired females
F0 females: 10 weeks prior to mating, during mating and during lactation period until sacrifice after weaning of their pups (PND26)
F1: selected at PND21-25, exposure occured at the same doses and schedule as their parents
Frequency of treatment:
7 days/week
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
- Age at mating of the mated animals in the study: 13-15 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 500, 5000 ppm
Basis:
nominal in diet
for actual doses see table1 under any other information on materials and methods including tables
No. of animals per sex per dose:
parental and F1: 24 males and females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a dose-range finding study
- Rationale for animal assignment: by stratified random sampling based on body weight

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all F0 and F1 parental rats were observed at least twice a day for general appearance and behavior. Dams were checked 3 times daily on days 21-25 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through the study. For dams, body weight was recorded weekly until evidence of copulation was detected and then on gestational days 0, 7, 14 and 20 and days 0, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined once a week and mean daily diet consumption calculated as g food/ day: Yes
Time schedule for examinations: weekly. For dams, food consumption was recorded through the exposure period, except during cohabitation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice a week, through the exposure period, except during cohabitation.
Oestrous cyclicity (parental animals):
Daily vaginal lavage samples were evaluated for each female for estrous cyclicity throughout the last 2 weeks before the cohabitation period and during cohabitation until evidence of copulation was detected. The presence of sperm in the vaginal smear and/ or a vaginal plug was considered as evidence of successful mating, and the day of successful mating was designated as day 0 of gestation.
Sperm parameters (parental animals):
Sperm parameters were determined in all F0 and F1 adult males on the day of sacrifice; The right testis was used to count testicular homogenization-resistant spermatid heads using a hemacytometer; The right epididymal cauda was weighed and used for sperm analysis (sperm number, sperm motility and morphology; Caudal sperm numbers were enumerated using a hemacytometer under a light microscope; For sperm motility (percentage of motile sperm and progressively motile sperm, swimming speed and pattern) were determined using a computer-assisted cell motion analyzer (TOX IVOS; Hamilton Thorne Bioscience, Beverly, MA, USA); Sperm morphology was evaluated for 200 sperm (stained with eosin and mounted on a slide glass) per male under a light microscope.
Litter observations:
STANDARDISATION OF LITTERS:
Performed on day 4 postpartum: Yes; maximum of 8 pups /litter (4/sex/litter), randomly selected.

PARAMETERS EXAMINED:
The following parameters were examined in all pups from F0 and F2 parents (F1 and F2 litters): number of pups, sex of pups, live birth, stillborn members per litter, and gross anomalies Clinical signs of toxicity (daily) and the body weight of live pups were measured on PND 0, 4, 7, 14 and 21.

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

OTHER:
Developmental landmarks:
- Pinna unfolding in all F1 and F2 live pups (for from PND 1 to PND 4); Eye opening beginning on PND12;
- The anogenital distance (AGD) was measured on PND 4 in all F1 and F2 pups using calipers, and the normalized value of AGD to body weight, AGD/cube root of the body weight ratio, was calculated (Gallavan et al., 1999);
- Incisor eruption beginning in one male and one female F1 and F2 pup selected from each dam were evaluated on PND 8 and eye opening beginning on PND 12, and continued until each pup achieved the criteria;
- The body weight of the respective F1 pups was recorded on the day the criteria were fulfilled.

Neuromotor performance:
- For the same F1 and F2 pups, surface righting reflex, negative geotaxis and mid-air righting reflex were assessed on PND 5, 8 and 18, respectively.

Sexual maturation:
- Preputial separation were observed daily for male in all F1 offspring selected as F1 parents beginning on PND 35 and
- Female vaginal opening were observed daily for female in all F1 offspring selected as F1 parents beginning on PND 25 until completion.
- The body weight of the respective F1 rats was recorded on the day of completion of these pubertal landmarks.

NEUROBEHAVIORAL EXAMINATIONS:
Locomotor activity:
- Spontaneous locomotor activity was measured in 10 male and 10 female F1 rats randomly selected from each group at 4weeks of age. A multi-channel activity monitoring system (SUPERMEX; Muromachi Kikai Co., Ltd., Tokyo, Japan) was employed.
Animals were placed individually in transparent polycarbonate cages [285(W)x 450(D) x 210(H) mm, CL-0108-1; CLEA Japan Inc., Tokyo, Japan], and spontaneous motor activity was measured using SUPERMEX (Muromachi Kikai Co., Ltd., Tokyo, Japan), which was placed above the cage to detect changes in heat across multiple zones of the cage with an infra-red sensor. Spontaneous motor activity was determined at 10-min intervals and for a total 60 min.

Spatial learning ability (T-maze test):
- Spatial learning ability was conducted using water-filled multiple T-maze test (Biel’s type) in 10 male and 10 female F1 rats selected from each group at 6 weeks of age.
The water temperature of the maze was maintained at 21.0–22° C. Each rat was allowed to swim three times in a straight channel on the day before the trial, and then tested in the maze with three trials per day for the following three consecutive days. The time required to reach the goal and the number of errors were recorded. To prevent the exhaustion, no animal was allowed to remain in the water for more than 3 min in any trial.
Postmortem examinations (parental animals):
SACRIFICE
- F0 and F1 parental male animals: after a parturition of their paired females;
- Maternal animals: were evaluated for estrous cycle stage by examination of the vaginal smear after weaning of pups, and euthanized in the proestrus stage by exsanguination under ether anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic, and abdominal viscera;
- The number of uterine implantation sites was recorded for each female;
- The testis and epididymis in males were prepared for microscopic examination and weighed;
- The brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididmydes, seminal vesicles (with coagulating glands and their fluids), ventral prostate, uterus and ovaries in males and females were weighed before fixation, fixed and underwent macroscopic examination; thyroid and seminal vesicles were weighed after fixation;
- The testis and epididymis were fixed with Bouin’s solution and preserved in 70% ethanol, and the other organs were stored in 10% neutral-buffered formalin.
- The number of primordial follicles in the right ovary was counted for 10 F1 females randomly selected from the control and highest dose groups. Every 20th section was mounted on a slide and stained with hematoxylin–eosin. About 40 sections per ovary were used to determine the primordial follicles.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluations were performed in F0 and F1 animals of the control and highest dose groups.

Of these animals, the testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina were embedded in paraffin ,sectioned, stained with hematoxylin–eosin and examined under a light microscope.
Postmortem examinations (offspring):
SACRIFICE
Non-parental F1 weanlings and all F2 offspring were euthanized under ether anesthesia at PND 26 of age.

HISTOPATHOLOGY / ORGAN WEIGTHS
For one male and one female F1 and F2 weanlings selected from each dam:
- the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries were removed, weighted and prepared for microscopic examination;
-Since test substance-related organ weight changes were found in the thymus, liver and spleen weight and in the liver and spleen weight of females in the highest dose group in F1 and F2 generations, they were histopathologically examined for the randomly selected 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups. Paraffin sections were routinely prepared, stained with hematoxyllin-eosin and examined using a light microscope.
Statistics:
Bartlett’s test for homogeneity of variances (p<0.05) was applied for homogeneity of distribution for parametric data (body weight, food and water consumption, length of estrous cycle and gestation, precoital interval, number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, parameters of behavioral tests, organ weight and sperm parameters);
For preweaning pups, body weight, AGD, viability, and age at completion of developmental landmarks were similarly analyzed using litter as experimental unit.
One way analysis of variance (p<0.10) was performed when homogeneity of distribution was established.
If a significant difference was detected, Dunnett’s test was conducted for comparisons between control and individual treatment groups (p<0.01 or 0.05).
Data without homogeneity were analyzed using Kruskal–Wallis rank sum test (p<0.10). If significant differences were found, Mann Whitney’s U test was conducted for comparison between control and each treatment group (p<0.01 or 0.05). Fisher’s exact test (p<0.01 or 0.05) was used to compare incidence of parental animals with clinical signs, and autopsy and histopathological findings, incidence of females with normal estrous cycles, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ratio and completion rate of negative geotaxis between AS and control group.
Wilcoxon rank sum test (p<0.01 or 0.05) was used to analyzed incidence of pups with clinical signs or autopsy findings per litter, completion rate of pinna unfolding in each litter, and success rate of surface and mid-air righting reflex.
Student’s t-test (p<0.01 or 0.05) was used to compare number of primordial follicles in control and highest dose groups because homogeneity of variance was indicated by F-test.
All of these statistical analyses were conducted using 5% level of probability as criterion for significance.
Reproductive indices:
The following reproductive indices for each exposed group were calculated in F0 and F1 generation parental animals:
- Copulation index (%) for males and females(no. of animals with successful copulation/no. of animals paired)×100;
- Precoital interval(days);
- Fertility index(%) for males and females (no. of animals that impregnated a female or were pregnant/no. of animals with successful copulation)×100;
- Gestation index (%)(no. of females that delivered live pups/no. of pregnant females)×100;
- Gestation length (days);
- Delivery index(%)(no. of pups delivered/no. of implantations)×100;
- Estrous cycle in F0 and F1 females.
Offspring viability indices:
For F1 and F2 offspring
Maternal indices:
No of litters;
No of pups delivered;
Sex ratio of pups total (no. of male pups/total no. of pups).

Viability index was calculated:
On PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered)×100;
On PND 4 (%) = (no. of live pups on PND 4/no. of live pups on PND 0)×100;
On PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) × 100.

Individual body weight:
Male and female individual weight during lactation on PND 0, 4, 7, 14 and 21.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
some effects in different live stages observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
some effects in different live stages observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: details provided in table1

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
during premating period a few AAS treated animals had persistent diestrous (not adverse)
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 and F1 males and females (50, 500 and 5000 ppm)
No treatment-related parental deaths or clinical signs of intoxication at any treatment groups in either male or female F0 and F1 rats.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
WATER CONSUMPTION
F0 males (50, 500 and 5000 ppm)
- was significantly lower during the entire 14-week treatment period in all three dose groups
F0 females (50ppm)
- was significantly lower than in controls during premating period (weeks 1,9 and 10 of dosing), 1 week of gestation and week 1 of lactation);
(500ppm)
- was significantly lower than in controls during premating, gestation and lactation period;
(5000 ppm)
- was significantly lower than in controls during premating, gestation and lactation period.
F1 males (50ppm)
- no significant changes were found compared to the control animals.
(500 and 5000 ppm)
- significantly lower than in controls throughout the treatment period
F1 females (50 ppm)
- was significantly lower than in controls during week 4 and 8-10 weeks of premating period;
(500, 5000 ppm)
- was significantly lower than in controls during premating, gestation and lactation period;

FOOD CONSUMPTION
F0 males (5000 ppm)
- was decreased in the 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F0 females (500 ppm)
- significantly decreased during week 1 of premating period.
(5000 ppm)
- significantly decreased at 1 week of premating period, and 2-3 weeks of lactation period.
F1 females (5000 ppm)
- significantly decreased at 2-3 weeks of lactation period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F1 males (50, 500 and 5000 ppm)
- no significant changes were observed in the Al treated and control groups.

BODY WEIGHT
F0 males (5000 ppm)
- significantly decreased in the first 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F0 females (5000 ppm)
- significantly decreased in the first 1 week of premating period and at the end of lactation period (21 day).
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F1 males (5000 ppm)
- significantly decreased in the first 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
F1 females (5000 ppm)
- significantly decreased in the first 1-2 weeks of premating period;
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test compound was administered with drinking water. The mean aluminium ammonium sulfate and elemental Al intakes during the whole dosing period in F0 males, F0 females, F1 males and F1 females provided in Table 1 (any other information on materials and methods including tables).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0 and F1 females (50, 500 and 5000 ppm)
estrous cycle
- during a premating period, a few AAS-treated F0 and F1 rats had persistent diestrus, however:
- no significant changes in the incidence of F0 and F1 females with normal estrous cycle (4-5 days) compared to the control animals were observed during the premating period;
- no significant differences in the estrous cycle between AAS treated and control groups were noted.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0 and F1 males (50, 500 and 5000 ppm)
There were no significant differences in the number of testis sperm and cauda epidymal sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm between control and AAS-treated groups in either F0 or F1 adults (however, no details were provided on the results of performed examination).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance
F0 and F1 parental animals
The authors reported that some animals failed to copulate, impregnate or deliver live pups, however:
- no significant differences were observed between the control and AAS-treated groups in either F0 and F1 generation (24 animals per group) for copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations, number of litters or pups delivered. Overall, there were no treatment-related effects on reproduction parameters.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Adults (F0 and F1)
- relative kidney weight was significantly increased in F0 females (500 and 5000ppm) and F1 males and females (5000ppm);
- absolute weight of the pituitary glands was significantly decreased in F0 females and F1 males and females (5000ppm);
- absolute thymus decreased significantly in F1 females(5000ppm);
- relative weight of seminal vesicle was significantly decreased in F1 males (50 ppm);
- absolute brain weight was significantly decreased in F1 females (500 ppm).

GROSS PATHOLOGY (PARENTAL ANIMALS)
F0 males and females, F1 males and females
No AAS treatment- related gross lesions were observed in either generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 males and females, F1 males and females
No treatment related histopathological changes of the reproductive organs were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
36.3 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: - decreased liver, spleen and thymus weights in the F1 and F2 males and females - decreased body weight gain in the F1 and F2 males and females - delay in sexual maturation in F1 females
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
NOAEL
Effect level:
5.41 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
bodyweight for F1 males and females (5000 ppm) decreased significantly, for F2 (5000ppm) lower body weight but not significantly
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
significantly delayed vaginal opening (F1 - 5000ppm)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see table3 + 4
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
F1 and F2 generation (50, 500 and 5000 ppm)
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation

CLINICAL SIGNS (OFFSPRING)
F1 generation
Clinical (external gross) examination during the preweaning period revealed microphthalmia, a rudimentary tail, trauma and scab on right hindlimb and crushing of incisor/malocclusion in a few F1 pups in control and AAS-treated groups (no data provided); however, there no significant differences in incidence between the control and AAS-treated animals were observed (data not shown);
F2 generation
No gross abnormalities in F2 pups were found in any groups.

BODY WEIGHT (OFFSPRING)
F1 generation (5000ppm group, F1 males and F1 females)
- body weight of male and female pups was significantly lower on PND 21 and on PND 14 and 21, respectively, compared to the control pups.
F2 generation (5000ppm, F2 males and females)
- body weights were lower than controls around time of weaning but no statistically significant differences were found.

SEXUAL MATURATION (OFFSPRING)
F1 females (5000 ppm)
vaginal opening
- was significantly delayed (32.3±1.8 days of age, compared to 30.2±2.1days of age in control). Body weight at the time of vaginal opening was slightly higher than the control (122.0±15.7 g. versus 115.8±12.6 g.) but no statistically significant difference was found.
F1 males (50, 500 and 5000 ppm)
the age at preputial separation
- no significant differences between control and AAS-treated groups were found and no changes were found in body weight at the time of completion of separation.

ORGAN WEIGHTS (OFFSPRING)
F1 males (Table 2) (5000 ppm)
Body weight was significantly decreased (85.97% compared to the control group); relative brain weight was significantly increased; absolute kidney weight was significantly decreased but the relative kidney weight was significantly increased ; absolute and relative thymus weight was significantly decreased;
absolute and relative liver weight was significantly decreased; absolute and relative spleen weight was significantly decreased; absolute weight of the adrenal glands was decreased significantly; absolute weight of testis and epididymis was significantly decreased.

F1 females (Table 3) (5000 ppm)
Body weight was significantly decreased (87.59% compared to the control group); relative brain weight was significantly increased; relative kidney weight was significantly increased; absolute thymus weight was significantly decreased; absolute liver weight was significantly decreased; absolute and relative spleen weight was significantly decreased; absolute weight of the adrenal glands was decreased significantly; absolute weight of uterus was significantly decreased.

F1 females (Table 3) (500 ppm)
weight of the adrenal glands was decreased significantly.

F2 males (Table 2) (5000 ppm)
Body weight was significantly decreased (92.21% compared to the control group); relative brain weight was significantly increased; absolute and relative thymus weight was significantly decreased; absolute liver weight was significantly decreased; relative kidney weight was significantly increased; absolute and relative spleen weight was significantly decreased.

F2 females (Table 3) (5000ppm)
Body weight was significantly decreased (90.50% compared to the control group); relative brain weight was significantly increased; absolute thymus weight was significantly decreased; absolute and relative liver weight was significantly decreased; relative kidney weight was significantly increased; absolute and relative spleen weight was significantly decreased; relative weight of the adrenal glands was increased significantly; absolute ovary weight was significantly decreased; absolute uterus weight was significantly decreased.

F1 females (5000ppm)
Number of primordial follicles in the ovary was no different between Al treated and control females (data not shown).

GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations
F1 males and females, F2 males and females
Gross observations did not reveal any compound-related lesions either in F1 and F2 weanlings or in pups found dead during the lactation period (no data provided that any pups were found dead during the lactation period).

HISTOPATHOLOGY (OFFSPRING)
F1 males and females, F2 males and females
No dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings and of the thymus in males in both generations.

OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT LANDMARKS
PHYSICAL DEVELOPMENT
F1 males/females and F2 males/females (50, 500 and 5000 ppm)
- Completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening , the AGD and AGD per cube root of the body weight ratio were not significantly different between the control and AAS-treated groups (data not shown).

NEUROMOTOR DEVELOPMENT
F1 males/females and F2 males/females (50, 500 and 5000 ppm)
- No significant changes were observed in the achieved day of the surface righting reflex (PND5), negative geotaxis reflex (PND8) and midair righting reflex (PND 18).
- No significant changes were observed in the response time of surface righting and negative geotaxis reflex (data not shown).

BEHAVIOR PERFORMANCE
SPONTANEOUS LOCOMOTOR ACTIVITY
F1 males (50, 500 and 5000 ppm)
- was not significantly different between control and AAS treated males at 10-min intervals and for 60 min;
F1 females (500 ppm)
Spontaneous locomotor activity was significantly decreased during the 40-50 and 50-60 minutes after start of test but no significant differences were observed in total activity for 60 min; no changes in spontaneous locomotor activity for 10 min intervals or for a total of 60 min between the control and the other AAS-treated groups in females

T MAZE TEST
Learning and memory performance in T-maze test (pre-test swimming trials in the straight channel)
F1 males and females (50, 500 and 5000 ppm)
- no significant changes were observed in the elapsed time to traverse the straight channel.
- no significant changes were observed in the elapsed time and number of errors on days 2–4.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table1: Absolute and relative organ weight of F1 and F2 male weanlings (% to the control)

AAS (ppm)   0     50     500     5000   
Organ weight  F1 males  F2 males  F1 males  F2 males  F1 males  F2 males  F1 males  F2 males
 number of animals  24  22  20  18  23  22  24  23
 body weigth (g)  100%  100%          85.97**  82.21*

 brain                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  113.59**  107.36*

 thymus                        

 absolute weigth (g)  100%  100%  NS  NS  NS  NS  76.79**  78.93**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  89.21*  86.09**

 liver                        

 absolute weight (g)  100%  100% NS NS   NS  NS  81.48**  89.56*
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  94.76*  NS

 kidneya                        

 absolute weigth (g)  100%  100%  NS  NS  NS  NS  90.74*  NS
rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  105.22*  107.96**

 spleen 

 
 absolute weight (g)  100%  100%  NS  NS  NS  NS  69.36**  74.86**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  80.76**  80.92**

 adrenala                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  90.91*  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

 testisa                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  90.02*  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

 epididymisa                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  84.01**  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

NS- no statistically significant differences were observed in the effect between Al treated and control animals

**- significantly different from the control, p<0.01; *- significantly different from the control, p<0.05;

a- value represent the total weights of the organs on both sides

Table2: Absolute and relative organ weights of F1 and F2 female weanlings (% to the control group)

AAS (ppm)   0   50     500     5000   
 organ weight  F1 females  F2 females  F1 females  F2 females  F1 females  F2 females  F1 females  F2 females
 number or animals  24  22  21  18  23  22  24  23
 body weight (g)  100%  100%          87.59**  90.50**

brain     

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  110 .88**  108.21**

 thymus      

 absolute weight (g)  100%  100%  NS  NS  NS  NS  82.72**  81.74**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

liver    

 absolute weight (g)  100%  100%  NS  NS  NS  NS  86.54**  84.73**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  93.49**

 kidneya   

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  107.96**  107.08

 spleen 

 absolute weight (g)  100%  100%  NS  NS  NS  NS  75.14**  77.75**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  85.57**  86.06**

 adrenala

 absolute weight (g)  100%  100%  NS  NS  89.01**  NS  87.45**  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS    NS  NS  109.45*

 ovarya

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  87.83**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

 uterus

 absolute weight (g)  100%  100%  NS  NS  NS  NS  74.89**  75.35*
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

NS- no statistically significant differences were observed in the effect between Al treated and control animals

**- significantly different from the control, p<0.01; *- significantly different from the control, p<0.05;

a- value represents the total weights of the organs on both sides

Applicant's summary and conclusion

Conclusions:
The major findings reported by the authors were decreased body weight in preweaning animals, delayed maturation of the female offspring, and decreased organ weight in the offspring. However, because the effects could be related to decrease fluid consumption and limited nursing ability of dams, the utility of this study for risk assessment is limited.
Limited examination of the internal and external abnormalities, morphological variations and malformations precludes rigorous assessment of the teratogenic potential of the AAS test compound. As urinanalysis was not performed, possible adverse effects of prolonged AAS ingestion on kidney function cannot be assessed.
Executive summary:

Hirata-Koizumi et al. (2011b) investigated the potential reproductive toxicity of aluminium ammonium sulfate (CAS#: 7784-25-0 (anhydrous)) CAS#: 7784-26-1 (dodecahydrate)] in a GLP and OECD TG 416 -compliant 2 generation reproductive toxicity study. 

Aluminium ammonium sulphate (AAS) was dissolved in deionized water at 0, 50, 500 or 5000 mg/L. The Al concentration in the deionized water was < 5 µg/mL and the Al content of the diet was 22-29 mg/kg Groups of 24 male and 24 female Crl:CD (SD) rats (F0 generation) were given AAS in drinking water from 5 weeks of age for 10 weeks prior to mating, during mating and gestation, when the parental males were culled, and for the females through weaning. Litters were normalized to 8 pups on PND 4. At weaning, 24 males and 24 females were selected to serve as the F1 generation and they were given AAS in drinking water for 10 weeks prior to mating, during mating and gestation, and for the females through weaning, as for the F0 generation. Exposure of the F1 weanlings occurred at the same concentrations as those of their parents.

Spontaneous locomotor activity was assessed at 4 weeks of age in 10 male and 10 female randomly selected F1 pups per group. Rats were placed in transparent polycarbonate cages and observed using an infrared sensor. Observations were made at “10 minute periods and for a total of 60 minutes.” At 6 weeks of age a water-filled multiple T-maze was used to assess the spatial learning abilities of 10 male and 10 female F1 pups from each treatment group (selected randomly). Habituation of swimming ability was conducted by allowing the animals to swim three times in a straight channel the day before the trial. Testing was done in blocks of three trials on three consecutive days. Rats were restricted to 3 min in the water to prevent physical exhaustion, but whether any rats were excluded on the basis of their performance was not reported. The parameters recorded to assess performance included time from entry into the water, elapsed time to traverse the straight channel, reaching the “goal ramp” and the numbers of errors. 

Drinking water consumption was reduced at all concentrations compared to that of the concurrent controls. These reductions were clearly concentration-dependent and the reduction was significant at 500 and 5000 ppm in males and females of the F0 and F1 generations. These reductions were significant at 50 ppm in the F0 males and at some intervals during AAS exposure of the F0 and F1 females. 

A transient decrease in food consumption was observed in the 500 and 5000 ppm groups and in body weight in the 5000 ppm group. One F1 male in the 500 ppm group died, but that death was not considered treatment-related.

There were no significant effects of AAS consumption on the oestrus cycle. The authors reported no differences for copulation, fertility index, gestation index, precoital interval, gestation length, number of implantations, live pups delivered or delivery index, sex ratios of pups or viability during the preweaning period in females compared to the control. There were no significant differences between control and AAS-treated groups regarding the numbers of testis and cauda epididymal sperm, percentage of motile and progressively motile sperm, sperm swimming patterns and speed or the numbers of morphologically-abnormal sperm. Moreover, there were no significant differences in the numbers of primordial follicles in the F1 ovaries between animals given 5000 ppm AAS and those consuming deionized water. 

In the F1 and F2 pups, there were no treatment-related differences in numbers of offspring with congenital malformations, sex-ratio or viability on PND 0, 4 or 21. Reduced body weights were reported in the F1 male and female pups at 5000 ppm, but not in lower dose groups. The F1 male pups had a significantly lower body weight on PND 21, F1 female pups on PND 14 and 21, and F2 male and female pups on the PND 26. In female F1 pups, vaginal opening was delayed significantly among those whose mothers consumed 5000 ppm AAS (mean ± S.D: 32.3 ± 1.8 days vs. 30.2 ± 2.1 days in control), but their body weights were not significantly different from those of the concurrent control at the time of vaginal opening.

Absolute weights of testes and epididymis of the F1 and F2 male pups at 5000 ppm were lower than control. Absolute weights of the uterus were significantly lower in the F1 female pups, and absolute weights of the ovary and uterus were significantly lower in the F2 females. Histopathological examination revealed no treatment-related changes in the reproductive organs. The authors considered these findings secondary to the decreased body weights and attributed the reductions in growth and development of the offspring “to the astringent taste of AAS which would decrease the palatability of drinking water in the AAS-treated groups”.

Spontaneous locomotor activity was no different among F1 males from dams given AAS in drinking water and those whose mothers consumed deionized water alone. There was some variation in activity among the F1 females.

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

The authors identified a LOAEL of 5000 mg AAS/L for both parental toxicity and reproductive toxicity (based on reduced preweaning body weight gain in F1 male (at PND 21) and female (PND 14, 21) pups, delay in the vaginal opening in F1 female pups, potentially attributed to inhibition of growth and decreased organ weights in F1 and F2 male and female offspring). The suggested LOAEL level corresponds to 36.3 mg Al/kg bw per day (Table 1, p.6). The reported NOAEL is 500 mg AAS/L which corresponds to 5.35 mg Al/kg bw per day.