Registration Dossier

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 weeks; (F1) 3-3.5 wks
- Weight at study initiation: (P) Males: ca. 180 g; Females: 120 g; (F1) Males: 80 g; Females: 80 g
- Housing: housed individually, except for 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 (White Flake, Charles River Laboratories Japan, Inc., Yokohama, Japan)
- Diet (e.g. ad libitum): standard diet (CRF-1; Oriental Japan), ad libitium
- Water (e.g. ad libitum): drinking water, supplied with different solutions
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 36-59
- Air changes (per hour): 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 at least once every 6 days and kept in a cool place until serving. Fresh drinking water was served at least once every 4 days

VEHICLE
- Concentration in vehicle: 0, 120, 600 or 3000 ppm

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until succeful copulation or mating period of 2 weeks had elapsed
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear. The day of successful mating was designated as day 0 of gestation
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the study, the concentrations of AS 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 by high performance liquid chromatography. AS contained in the drinking water for the control group was less than the quantitation limit (5 µg/mL). Aluminium concentration in the standard diet, analyzed by atomic absorption spectrometry for each lot of diet, ranged from 25 ppm to 29 ppm.

Duration of treatment / exposure:

F0: Twenty-four F0 rats (5-week-old males and females)/sex/group were exposed to AS in drinking water at 0, 120, 600 or 3000 ppm. After 10-week administration of AS, each female rat was mated with a male rat of the same dosage group, and pregnant females were allowed to deliver spontaneously and nurse their pups. Administration of AS was continued throughout the mating, gestation and lactation periods. F0 parental male rats were necropsied after the parturition of paired females. F0 females were necropsied after weaning of their pups.
F1: 24 male and female weanlings were selected as F1 parents on postnatal day (PND) 21-25, which was day 0 of the 10-week prior mating dosing for the F1 generation. F1-selected rats were given drinking water with the respective formulation, and were mated, allowed to deliver and nurse their F2 pups, and necropsied in the same manner as described for F0 rats.
Unselected F1 weanlings and all F2 weanlings were necropsied on PND 26.

Frequency of treatment:

Continuously in drinking water

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: F0: 15 weeks, F1: 13 weeks

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Prior to the present two-generation reproductive toxicity study, a dose-finding study was performed in male and female rats given drinking water containing AS at 0, 1000, 3000, 10000 or 30000 ppm. In that study, males were dosed for 7 weeks, beginning 14 days before mating, and females were dosed for 6–8 weeks beginning 14 days before mating to day 4 of lactation throughout the mating and gestation period.
In the highest dose group, animals were euthanized at the end of the 2nd week of administration because of a marked decrease in body weight as a result of water avoidance. Water consumption also decreased in all other treatment groups. Decreased food consumption and body weight were observed at 3000 ppm and above. At autopsy, thickening of the limiting ridge in the stomach, and atrophy of the thymus and spleen were detected at 10000 ppm. The relative weights of theliver, thymus and spleen were decreased in females in 3000 and 10000 ppm groups.
Although there were no changes in any reproductive parameters, the body weights of pups on postnatal day (PND) 4 were decreased at 10000 ppm. Taking into account the results of this dose-finding study, the dose levels of AS in the present study were set as 120, 600 or 3000 ppm.

Positive control:

N.A.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; for females exhibiting evidence of succefsul mating body weight was recorded on gestational days 0, 7, 14 and 20 and days 0, 4, 7, 14 and 21 of lactation

FOOD CONSUMPTION: Yes
- Time schedule for examination: weekly, or females exhibiting evidence of succesful mating food consumptiont was recorded on gestational days 0, 7, 14 and 20 and days 0, 7, 14 and 21 of lactation
- Food consumption calculated as g/day

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was recorded twice a week, and on days 0, 4, 7, 11, 14, 17 and 20 of gestation and days 0, 4, 7, 11, 14, 17, 19 and 21 of lactation. The intake of test substance was calculated based upon mean values for body weight and water consumption in each group.

Oestrous cyclicity (parental animals):

For each female, daily vaginal lavage samples were evaluated for estrous cyclicity throughout the last 2 weeks of the premating period and during cohabitation until evidence of copulation was detected. Females having repeated 4–6 day estrous cycles were judged to have normal estrous cycles.

Sperm parameters (parental animals):

Parameters examined in all F0 and F1 male parental generations:
The 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 computer-assisted cell motion analyzer (TOX IVOS; Hamilton Thorne Bioscience, Beverly, MA, USA). After recording sperm motion, the cauda epididymal fluid was diluted and the sperm were enumerated with a hemacytometer under a light microscope. Sperm count per gram of epididymal tissue was obtained by dividing the total count by the gram weight of the cauda epididymis. The sperm was stained with eosin and mounted on a slide glass. Two hundred sperm in each sample were examined under a light microscope, and the percentage of morphologically abnormal sperm was calculated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, 8 pups/litter (4/sex/litter); no adjustment was made for litters of fewer than eight pups

PARAMETERS EXAMINED
The following parameters were examined in F0 and F1 offspring:
Number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain (body weight was recorded on days 0, 4, 7 , 17 and 21), daily for clinical signs of toxicity,
other: Pinna unfolding from PND 1 to 4 (during with body weight was recorded daily), the anogenital distance (AD) was measured using calipers on PND and the normalized value of AGD to body weight. AGD/cube root of the body weight ratio, was calculated. One male and one female F1 and F2 pup selected from each dam were evaluated for incisor eruption beginning on PND 8 and eye opening beginning on PND 12, and
continued until each pup fulfilled the criteria. The body weight of the respective F1 and F2 pups was recorded on the day the criteria were fulfilled. Surface righting reflex, negative geotaxis and mid-air righting reflex were assessed on PND 5, 8 and 18, respectively, for one male and one female F1 and F2 pup selected from each dam. All F1 offspring selected as F1 parents were observed daily for male preputial separation beginning on PND 35 or female vaginal opening 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.

Behavioral test:
Spontaneous locomotor activity was measured at 4 weeks of age in 10 male and 10 female F1 rats randomly selected from each group, using a multi-channel activity monitoring system (SUPERMEX; Muromachi Kikai Co., Ltd., Tokyo, Japan). Rats were placed individually in transparent polycarbonate cages [285 (W) mm×450 (D) mm×210 (H) mm, CL-0108-1; CLEA Japan, Inc., Tokyo, Japan], which were placed under an infrared sensor that detects thermal radiation from animals, and spontaneous motor activity was determined at 10-min intervals and for 60 min.
A test in a water-filled multiple T-maze was conducted in 10 male and 10 female F1 rats selected from each group at 6 weeks of age. The water temperature of the maze was kept 20.5–22 °C. As a preliminary swimming ability test, each rat was allowed to swim three times in a straight channel on the day before the maze trial, and then tested in the maze with three trials per day for the next three consecutive days. The elapsed time between entry into the water at the starting point and touching the goal ramp, and thenumber of errors were recorded. To prevent the exhaustion of the rats, no animal was allowed to remain in the water for more than 3min in any trial.

OTHER:
Following the adjustment of litter size on PND4, culled pups were euthanized by inhalation of carbon dioxide and subjected to a gross external and internal observation. Grossly abnormal organs/tissues were removed and stored in 10 % neutral-buffered formalin. All pups found dead before weaning were necropsied immediately, and the whole body was stored in 10 % neutral-buffered formalin.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were euthanized by exsanguination under ether anesthesia after the parturition of paired females.
- Maternal animals: All surviving animals showing successful reproductive performance were evaluated for estrous cycle stage by examination of the vaginal smear after weaning of pups, and euthanized at the proestrous stage by exsanguination under ether anesthesia. Females that did not copulate or had not completed parturition and dams with total litter loss were euthanized in the same way around the same time as females with successful reproduction.

GROSS NECROPSY
- For all parental animals, the external surfaces were examined. The abdomen and thoracic cavity were opened, and gross internal examination was performed. Major organs were removed and the number of uterine implantation sites was recorded for each female. 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 brain, pituitary, thyroids, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, seminal vesicles (with coagulating glands and their fluids), ventral prostate, uterus and ovaries were weighed before fixation. The thyroid and seminal vesicle were weighed after fixation.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluations were performed in all animals of the control and highest dose groups, in females with abnormal estrous cycles, abnormal delivery or totally dead pups, in males and females without evidence of copulation or insemination, and in all animals with grossly abnormal reproductive organs. Of these animals, the testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina, which were fixed as mentioned above, were embedded in paraffin by a routine procedure. They were sectioned, stained with hematoxylin–eosin and examined histopathologically under a light microscope. If treatment-related histopathological changes were found in the highest dose group, were the same tissues from the next lower dose group then examined.

OTHER:
In 10 F1 females, randomly selected from the control and highest dose groups, the number of primordial follicles was counted as follows. The right ovary, fixed in 10 % neutral-buffered formalin, was dehydrated and then embedded in paraffin paraffin in longitudinal orientation by routine procedures. Sections were cut serially at 5 µm and every 20th section was serially mounted on a slide and stained with hematoxylin and eosin. About 40 sections per ovary were used to determine the primordial follicles.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 26 days of age.
- These animals were subjected to postmortem examinations as described for the adults.
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 the organ weights were measured. Major organs, including the weighed organs, were stored in 10 % neutral-buffered formalin.

HISTOPATHOLOGY
Since test substance-related organ weight changes were found in the liver and spleen of the highest dose group, they were histopathologically examined for 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups. The examined animals were randomly selected from animals whose organs were stored. If treatment-related histopathological changes were observed in the highest dose group, were the same tissues from the next lower dose group then examined. For the histopathological examination, paraffin sections were routinely prepared and stained with hematoxylin and eosin.

Statistics:

Parametric data, such as body weight, food and water consumption, length of the estrous cycle and gestation, precoital interval, the number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, parameters of behavioral tests, organ weight and sperm parameters, were analyzed by Bartlett’s test for homogeneity of distribution.
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.
When homogeneity was recognized, oneway analysis of variance was performed. If a significant difference was detected, Dunnett’s test was conducted for comparisons 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 Whitney’s U test was conducted for comparison between the control and each dosage group.
The incidence of parental animals with clinical signs, and autopsy and histopathological findings, the 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 were compared between the AS and control group using Fisher’s exact test.
The incidence of pups with clinical signs or autopsy findings per litter, the completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex were analyzed by the Wilcoxon rank sum test.
The number of primordial follicles in the control and highest dose groups was compared by Student’s t-test because the homogeneity of variance was indicated by the F-test.
All of these statistical analyses were conducted using the 5 % level of probability as the criterion for significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

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: see below

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- 120 ppm group: 1 F1 male was found dead at 9 weeks of dosing. In this animal, soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. At autopsy, various changes, including accumulation of ascitic and pleural fluid and dark purple discoloration of the liver and kidneys, were found.
- 600 ppm group: 1 F0 female was found dead at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this animal from the beginning of 5 weeks of dosing. No abnormality was found on gross interal examination.
- 3000 ppm group: 1 F1 male was found dead at 12 weeks of dosing without any clinical sign of toxicity. No abnormality was found on gross internal examination.
No significant difference was seen between control and AS-treated groups in the incidence of clinical signs of toxicity in either male or female F0 and F1 rats (No data shown).

BODY WEIGHT, WATER AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0:
In F0 males and females of all AS-treated groups, water consumption was significantly lower than in controls almost throughout the dosing period. In F0 males, there were significant decreases in food consumption in the first week of dosing at 600 and 3000 ppm, and during week 8 and weeks 13–14 of dosing at 3000 ppm. Food consumption of F0 females showed a significantly lower value during week 1 of dosing at 3000 ppm and during week 3 of lactation at 600 and 3000 ppm. The body weight of F0 males and females was significantly lowered in the first 2 or 3 weeks of dosing at 3000 ppm.

F1:
Water consumption was significantly decreased through the dosing period in 600 ppm and 3000 ppm treated males, and during weeks 3–6, week 8 and week 10 of dosing in 120 ppm treated males. In F1 females, significant reductions in water consumption were found almost throughout the dosing period at 3000 ppm, during week 10 of dosing and week 3 of lactation at 600 ppm, and during weeks 9–10 of dosing at 120 ppm. Food consumption was significantly decreased during week 10 of dosing in F1 males of the 600 and 3000 ppm groups, and during week 3 of lactation in F1 females of the same groups. There was also a transient significant increase in food consumption during week 6 of dosing in F1 females of the 120 ppm group. The body weight of F1 males and females exhibited no significant differences between the control and AS-treated groups, except that F1 females of the 120 ppm group had significantly higher body weight during weeks 6–8 of dosing.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on water consumption and body weight, daily AS intakes during the premating and postmating periods in males and during the premating, gestation and lactation periods in females were calculated for each of the AS-treated groups.
Calculated mean AS intakes during the whole of these period were:
8.6, 41.0 and 188 mg/kg bw/day in F0 males,
14.4, 71.5 and 316 mg/kg bw/day in F0 females,
10.7, 50.2 and 232 mg/kg bw/day in F1 males, and
15.3, 74.2 and 338 mg/kg bw/day in F1 females,
in the 120, 600 and 3000 ppm groups, respectively.

The total ingested dose of aluminium from drinking water and food combined was estimated from the water and food consumption and body weight. Average aluminium intake was
1.62, 2.96, 8.06 and 31.2 mg Al/kg bw/day in F0 males,
2.29, 4.50, 13.5 and 52.0 mg Al/kg bw/day in F0 females,
1.93, 3.55, 9.78 and 38.5 mg Al/kg bw/day in F1 males, and
2.35, 4.72, 14.0 and 55.6 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, AS produced no significant deviations in the estrous cycle of F0 and F1 females although 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 (data not shown).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm parameters examined for scheduled sacrificed adults, in F0 generation, the absolute number of cauda epididymal sperm was significantly decreased at 3000 ppm (253.8±61.3×10^6/cauda versus 286.3±40.3×10^6/cauda in the control); however, no significant changes were found in the number per gram of tissue. No such change was observed in F1 adults. There were no significant differences in the 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 between control and AS-treated groups in either F0 or F1 adults (data not shown).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
During the mating period, copulation was not observed in two males each in the control, 120 ppm and 3000 ppm groups and in one female of the control group in the F0 generation. In the F1 generation, one male in the control group, two males and one female in the 120 ppm group, one male in the
600 ppm group, and three males and one female in the 3000 ppm group did not copulate. Among females with successful copulation, one female each in the control and 3000 ppm group and two females at 120 ppm in the F0 generation and two females each in the control, 600 ppm and 3000 ppm groups, and four females at 120 ppm in the F1 generation were not impregnated. In addition, one pregnant F0 female each at 120, 600 and 3000 ppm and one pregnant F1 female at 120 ppm did not deliver live pups; however, there were no significant differences in the copulation, fertility or gestation index, and the precoital interval or gestation length between the control and AS-treated groups in F0 and F1 generation. No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 males, absolute and relative liver weights were significantly decreased at 3000 ppm. Absolute spleen weight was also decreased significantly in this group, but no significant change was found in the relative weight.
In F1 males, the absolute weights of the adrenals at 3000 ppm and the testes at 600 ppm were significantly decreased without significant changes in the relative weight.
There were no significant changes in the absolute and relative weights of any organ in F0 and F1 female adults (data not shown).

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

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of the reproductive organs revealed no compound-related alterations. There was no significant difference in the number of primordial follicles in the ovary of F1 females between control and 3000 ppm groups (data not shown).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see below

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

see below

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- 120 ppm group: 1 F1 male was found dead at 9 weeks of dosing. In this animal, soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. At autopsy, various changes, including accumulation of ascitic and pleural fluid and dark purple discoloration of the liver and kidneys, were found.
- 600 ppm group: 1 F0 female was found dead at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this animal from the beginning of 5 weeks of dosing. No abnormality was found on gross interal examination.
- 3000 ppm group: 1 F1 male was found dead at 12 weeks of dosing without any clinical sign of toxicity. No abnormality was found on gross internal examination.
No significant difference was seen between control and AS-treated groups in the incidence of clinical signs of toxicity in either male or female F0 and F1 rats (No data shown).

BODY WEIGHT, WATER AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0:
In F0 males and females of all AS-treated groups, water consumption was significantly lower than in controls almost throughout the dosing period. In F0 males, there were significant decreases in food consumption in the first week of dosing at 600 and 3000 ppm, and during week 8 and weeks 13–14 of dosing at 3000 ppm. Food consumption of F0 females showed a significantly lower value during week 1 of dosing at 3000 ppm and during week 3 of lactation at 600 and 3000 ppm. The body weight of F0 males and females was significantly lowered in the first 2 or 3 weeks of dosing at 3000 ppm.

F1:
Water consumption was significantly decreased through the dosing period in 600 ppm and 3000 ppm treated males, and during weeks 3–6, week 8 and week 10 of dosing in 120 ppm treated males. In F1 females, significant reductions in water consumption were found almost throughout the dosing period at 3000 ppm, during week 10 of dosing and week 3 of lactation at 600 ppm, and during weeks 9–10 of dosing at 120 ppm. Food consumption was significantly decreased during week 10 of dosing in F1 males of the 600 and 3000 ppm groups, and during week 3 of lactation in F1 females of the same groups. There was also a transient significant increase in food consumption during week 6 of dosing in F1 females of the 120 ppm group. The body weight of F1 males and females exhibited no significant differences between the control and AS-treated groups, except that F1 females of the 120 ppm group had significantly higher body weight during weeks 6–8 of dosing.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on water consumption and body weight, daily AS intakes during the premating and postmating periods in males and during the premating, gestation and lactation periods in females were calculated for each of the AS-treated groups.
Calculated mean AS intakes during the whole of these period were:
8.6, 41.0 and 188 mg/kg bw/day in F0 males,
14.4, 71.5 and 316 mg/kg bw/day in F0 females,
10.7, 50.2 and 232 mg/kg bw/day in F1 males, and
15.3, 74.2 and 338 mg/kg bw/day in F1 females,
in the 120, 600 and 3000 ppm groups, respectively.

The total ingested dose of aluminium from drinking water and food combined was estimated from the water and food consumption and body weight. Average aluminium intake was
1.62, 2.96, 8.06 and 31.2 mg Al/kg bw/day in F0 males,
2.29, 4.50, 13.5 and 52.0 mg Al/kg bw/day in F0 females,
1.93, 3.55, 9.78 and 38.5 mg Al/kg bw/day in F1 males, and
2.35, 4.72, 14.0 and 55.6 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, AS produced no significant deviations in the estrous cycle of F0 and F1 females although 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 (data not shown).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm parameters examined for scheduled sacrificed adults, in F0 generation, the absolute number of cauda epididymal sperm was significantly decreased at 3000 ppm (253.8±61.3×10^6/cauda versus 286.3±40.3×10^6/cauda in the control); however, no significant changes were found in the number per gram of tissue. No such change was observed in F1 adults. There were no significant differences in the 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 between control and AS-treated groups in either F0 or F1 adults (data not shown).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
During the mating period, copulation was not observed in two males each in the control, 120 ppm and 3000 ppm groups and in one female of the control group in the F0 generation. In the F1 generation, one male in the control group, two males and one female in the 120 ppm group, one male in the
600 ppm group, and three males and one female in the 3000 ppm group did not copulate. Among females with successful copulation, one female each in the control and 3000 ppm group and two females at 120 ppm in the F0 generation and two females each in the control, 600 ppm and 3000 ppm groups, and four females at 120 ppm in the F1 generation were not impregnated. In addition, one pregnant F0 female each at 120, 600 and 3000 ppm and one pregnant F1 female at 120 ppm did not deliver live pups; however, there were no significant differences in the copulation, fertility or gestation index, and the precoital interval or gestation length between the control and AS-treated groups in F0 and F1 generation. No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 males, absolute and relative liver weights were significantly decreased at 3000 ppm. Absolute spleen weight was also decreased significantly in this group, but no significant change was found in the relative weight.
In F1 males, the absolute weights of the adrenals at 3000 ppm and the testes at 600 ppm were significantly decreased without significant changes in the relative weight.
There were no significant changes in the absolute and relative weights of any organ in F0 and F1 female adults (data not shown).

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

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of the reproductive organs revealed no compound-related alterations. There was no significant difference in the number of primordial follicles in the ovary of F1 females between control and 3000 ppm groups (data not shown).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
See table 2
No significant changes were found in the sex ratio of pups and the viability index in either generation.

CLINICAL SIGNS (OFFSPRING)
For the physical development of male and female F1 pups and male F2 pups, there was no significant difference in the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening between the control and AS-treated groups. In female F2 pups, the completion rate of pinna unfolding on PND 2 was significantly lower in the 600 ppm group (17.0±35.4 %, compared with 45.8±46.9 in controls), but no dose dependency was observed in this change. No significant changes were found in the completion rate of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks in female F2 pups. 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).
All male and female F1 pups in all groups achieved the 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 time of surface righting and negative geotaxis reflex. In F2 pups, one female of the 600 ppm group failed in one of three trials of the mid-air righting reflex on PND 18; however, there was no significant difference in the mean success rate between the control and 600ppm groups (100±0.0 % versus 98.4±7.3 %). The 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, and no significant changes were found in the response time (data not shown).

BODY WEIGHT (OFFSPRING)
See table 2
In the 3000 ppm group, the body weight of male and female F1 pups was significantly lower than the control on PND 21. Body weights of F2 female pups were also significantly lower than controls on PND 21 at 3000 ppm. There were no significant differences in the body weight of male F2 pups between the control and AS-treated groups during the preweaning period.

SEXUAL MATURATION (OFFSPRING)
As for the sexual development of F1 male and female animals, vaginal opening was significantly delayed at 3000 ppm (31.4±1.7, compared to 29.5±2.1 in control). At this dose, body weight at the time of vaginal opening was slightly heavier than the control (119.0±13.3 g versus 109.6±11.6 g) although not statistically significant.
No significant differences between control and AS-treated groups were noted in the age at preputial separation or body weight at the time of completion in males.

ORGAN WEIGHTS (OFFSPRING)
F1: See table 3
The 3000 ppm treated males and females had a significantly lower body weight at scheduled sacrifice than the controls. In this group, absolute and relative liver weights were significantly lower than the controls. Absolute spleen weight was also decreased significantly in both sexes of the 3000 ppm group, accompanied by a significant decrease in the relative weight in males. In addition, significant decreases in the absolute weight were found for the thymus in both sexes and for the kidneys, testes and epididymides in males at 3000 ppm, and for the uterus in females at 600 and 3000 ppm. Relative brain weight was significantly increased in both sexes of the 3000ppm group.
F2: See table 4
The mean body weight at scheduled sacrifice was significantly lowered in both sexes of the 3000 ppm group. In males, the absolute and relative weights of the thymus and spleen were significantly decreased in the 3000 ppm group. Significant decreases were also found in the absolute weight of the liver and epididymides at 3000 ppm. The relative brain weight was significantly increased at this dose. At 120 ppm, the only significant change was a non-dose-related decrease in the relative thymus weight. In F2 females, there were significant decreases in the absolute and relative weights of the liver, and the absolute weight of the spleen, ovary and uterus, and a significant increase in the relative brain weight at 3000 ppm. In addition, a significant decrease in the absolute brain weight was observed only in the 600 ppm group.

GROSS PATHOLOGY (OFFSPRING)
Gross examination of delivered pups revealed one F1 pup with trauma in the perianal region and tail in the control group and one F1 pup with hemimelia and oligodactyly in the 120 ppm group, but no significant difference was found in the incidence between the control and AS-treated groups. No malformed F2 pups were found in any groups.
External and internal gross observations revealed no compound-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period.

HISTOPATHOLOGY (OFFSPRING)
There were no dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings.

OTHER FINDINGS (OFFSPRING) - Behavioral effects:
Spontaneous locomotor activity at 10 min intervals and for 60 min was not significantly different between control and AS treated groups in male and female F1 rats. In the water-filled T-maze test, pre-test swimming trials in the straight channel revealed that all male and female F1 rats in each group could swim satisfactorily, and no significant changes were observed in the elapsed time to traverse the straight channel. On days 2–4 of the T-maze test, no significant changes were observed in the elapsed time and number of errors in males. In females, the elapsed time and the number of errors on day 2 of the T-maze was significantly lowered at 600 ppm, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 of the T-maze test between control and AS-treated groups (data not shown).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
See table 2
No significant changes were found in the sex ratio of pups and the viability index in either generation.

CLINICAL SIGNS (OFFSPRING)
For the physical development of male and female F1 pups and male F2 pups, there was no significant difference in the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening between the control and AS-treated groups. In female F2 pups, the completion rate of pinna unfolding on PND 2 was significantly lower in the 600 ppm group (17.0±35.4 %, compared with 45.8±46.9 in controls), but no dose dependency was observed in this change. No significant changes were found in the completion rate of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks in female F2 pups. 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).
All male and female F1 pups in all groups achieved the 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 time of surface righting and negative geotaxis reflex. In F2 pups, one female of the 600 ppm group failed in one of three trials of the mid-air righting reflex on PND 18; however, there was no significant difference in the mean success rate between the control and 600ppm groups (100±0.0 % versus 98.4±7.3 %). The 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, and no significant changes were found in the response time (data not shown).

BODY WEIGHT (OFFSPRING)
See table 2
In the 3000 ppm group, the body weight of male and female F1 pups was significantly lower than the control on PND 21. Body weights of F2 female pups were also significantly lower than controls on PND 21 at 3000 ppm. There were no significant differences in the body weight of male F2 pups between the control and AS-treated groups during the preweaning period.

SEXUAL MATURATION (OFFSPRING)
As for the sexual development of F1 male and female animals, vaginal opening was significantly delayed at 3000 ppm (31.4±1.7, compared to 29.5±2.1 in control). At this dose, body weight at the time of vaginal opening was slightly heavier than the control (119.0±13.3 g versus 109.6±11.6 g) although not statistically significant.
No significant differences between control and AS-treated groups were noted in the age at preputial separation or body weight at the time of completion in males.

ORGAN WEIGHTS (OFFSPRING)
F1: See table 3
The 3000 ppm treated males and females had a significantly lower body weight at scheduled sacrifice than the controls. In this group, absolute and relative liver weights were significantly lower than the controls. Absolute spleen weight was also decreased significantly in both sexes of the 3000 ppm group, accompanied by a significant decrease in the relative weight in males. In addition, significant decreases in the absolute weight were found for the thymus in both sexes and for the kidneys, testes and epididymides in males at 3000 ppm, and for the uterus in females at 600 and 3000 ppm. Relative brain weight was significantly increased in both sexes of the 3000ppm group.
F2: See table 4
The mean body weight at scheduled sacrifice was significantly lowered in both sexes of the 3000 ppm group. In males, the absolute and relative weights of the thymus and spleen were significantly decreased in the 3000 ppm group. Significant decreases were also found in the absolute weight of the liver and epididymides at 3000 ppm. The relative brain weight was significantly increased at this dose. At 120 ppm, the only significant change was a non-dose-related decrease in the relative thymus weight. In F2 females, there were significant decreases in the absolute and relative weights of the liver, and the absolute weight of the spleen, ovary and uterus, and a significant increase in the relative brain weight at 3000 ppm. In addition, a significant decrease in the absolute brain weight was observed only in the 600 ppm group.

GROSS PATHOLOGY (OFFSPRING)
Gross examination of delivered pups revealed one F1 pup with trauma in the perianal region and tail in the control group and one F1 pup with hemimelia and oligodactyly in the 120 ppm group, but no significant difference was found in the incidence between the control and AS-treated groups. No malformed F2 pups were found in any groups.
External and internal gross observations revealed no compound-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period.

HISTOPATHOLOGY (OFFSPRING)
There were no dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings.

OTHER FINDINGS (OFFSPRING) - Behavioral effects:
Spontaneous locomotor activity at 10 min intervals and for 60 min was not significantly different between control and AS treated groups in male and female F1 rats. In the water-filled T-maze test, pre-test swimming trials in the straight channel revealed that all male and female F1 rats in each group could swim satisfactorily, and no significant changes were observed in the elapsed time to traverse the straight channel. On days 2–4 of the T-maze test, no significant changes were observed in the elapsed time and number of errors in males. In females, the elapsed time and the number of errors on day 2 of the T-maze was significantly lowered at 600 ppm, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 of the T-maze test between control and AS-treated groups (data not shown).

Table 1

Reproductive performance of F0 and F1 parental animals

AS (ppm)		0 (control)	120	600	3000
F0 generation
No. of rats (male/female)		24/24	24/24	24/24	24/24
Copulation index (%)^a	Males	91.7	91.7	100	91.7
	Females	95.8	100	100	100
Precoital interval (days)^b		3.2 ± 1.1	3.2 ± 1.8	2.9 ± 1.3	2.8 ± 1.6
Fertility index (%)^c	Males	95.5	90.9	100	95.5
	Females	95.7	91.7	100	95.8
Gestation index (%)^d		100	95.5	95.7	95.7
Gestation length (days)^b		22.4 ± 0.5	22.5 ± 0.6	22.1 ± 0.4	22.3 ± 0.5
Delivery index (%)^{b, e}		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 (%)^a	Males	95.8	91.3	95.8	87.5
	Females	100	95.8	100	95.8
Precoital interval (days)^b		3.3 ± 3.2	3.0 ± 2.0	2.7 ± 1.5	2.3 ± 1.1
Fertility index (%)^c	Males	91.3	81.0	91.3	95.2
	Females	91.7	82.6	91.7	91.3
Gestation index (%)^d		100	94.7	100	100
Gestation length (days)^b		22.4 ± 0.5	22.3 ± 0.5	22.2 ± 0.4	22.2 ± 0.4
Delivery index (%)^{b, e}		94.0 ± 9.9	87.5 ± 22.6	91.4 ± 10.7	94.6 ± 6.8

a Copulation index (%) = (no. of animals with successful copulation/no. of animals paired) × 100.

b Values are given as the mean ± S.D.

c Fertility index (%) = (no. of animals that impregnated a female or were pregnant/no. of animals with successful copulation) × 100.

d Gestation index (%) = (no. of females that delivered live pups/no. of pregnant females) × 100.

e Delivery index (%) = (no. of pups delivered/no. of implantations) × 100.

Table 2

Sex ratio, viability and body weight for F1 and F2 pups.

AS (ppm)	0 (control)	120	600	3000
F1 offspring
No. of litters	22	21	22	22
No. of pups delivered^a	13.9 ± 1.7	12.4 ± 4.7	13.1 ± 4.1	13.1 ± 3.4
Sex ratio of pups^b	0.503	0.462	0.513	0.536
Viability index of pups (%)^a
On PND 0^c	100.0 ± 0.0	99.3 ± 2.3	99.7 ± 1.6	99.5 ± 2.4
On PND 4^d	98.7 ± 2.9	95.2 ± 21.8	98.8 ± 2.6	98.0 ± 5.4
On PND 21^e	99.4 ± 2.7	100.0 ± 0.0	100.0 ± 0.0	99.4 ± 2.7
Male pup weight during lactation (g)^a
On PND 0	7.05 ± 0.61	7.25 ± 0.99	6.74 ± 0.69	6.96 ± 0.76
On PND 4	11.04 ± 0.85	11.41 ± 1.99	10.86 ± 1.37	11.00 ± 1.06
On PND 7	18.91 ± 1.29	19.36 ± 2.77	18.59 ± 1.71	18.47 ± 1.35
On PND 14	37.70 ± 2.63	37.97 ± 3.08	37.39 ± 2.59	36.34 ± 2.41
On PND 21	62.48 ± 4.50	62.63 ± 6.14	60.77 ± 4.01	57.34 ± 4.86**
Female pup weight during lactation (g)^a
On PND 0	6.61 ± 0.55	6.89 ± 0.83	6.35 ± 0.57	6.60 ± 0.64
On PND 4	10.46 ± 0.89	11.06 ± 1.71	10.27 ± 1.33	10.43 ± 0.83
On PND 7	18.03 ± 1.27	18.56 ± 2.31	17.69 ± 1.61	17.61 ± 1.21
On PND 14	36.29 ± 2.71	36.94 ± 3.03	35.67 ± 2.60	35.31 ± 2.24
On PND 21	60.17 ± 4.16	60.87 ± 5.68	57.68 ± 4.33	55.60 ± 4.34**
F2 offspring
No. of litters	22	18	22	21
No. of pups delivered^a	13.1 ± 3.6	13.2 ± 3.8	12.6 ± 3.9	14.0 ± 1.9
Sex ratio of pups^b	0.528	0.502	0.536	0.457
Viability index of pups (%)^a
On PND 0^c	99.68 ± 1.51	99.49 ± 2.14	98.42 ± 3.57	98.69 ± 3.60
On PND 4^d	94.72 ± 14.54	98.07 ± 5.45	99.07 ± 3.15	99.01 ± 2.49
On PND 21^e	100.00 ± 0.00	98.61 ± 4.04	100.00 ± 0.00	100.00 ± 0.00
Male pup weight during lactation (g)^a
On PND 0	6.97 ± 0.68	6.92 ± 0.81	6.87 ± 0.74	6.89 ± 0.60
On PND 4	10.73 ± 1.62	10.53 ± 1.27	11.27 ± 1.81	10.52 ± 1.15
On PND 7	17.96 ± 2.05	17.51 ± 2.12	18.83 ± 2.39	17.72 ± 1.60
On PND 14	35.79 ± 3.52	36.18 ± 3.63	37.32 ± 4.15	35.44 ± 2.73
On PND 21	59.61 ± 5.45	59.44 ± 5.67	60.12 ± 7.12	56.36 ± 4.47
Female pup weight during lactation (g)^a
On PND 0	6.66 ± 0.69	6.38 ± 0.78	6.41 ± 0.65	6.50 ± 0.49
On PND 4	10.22 ± 1.63	9.70 ± 1.23	10.36 ± 1.54	9.98 ± 0.91
On PND 7	17.03 ± 1.99	16.36 ± 2.35	17.40 ± 2.18	16.89 ± 1.23
On PND 14	34.82 ± 3.52	34.17 ± 3.58	34.96 ± 4.24	34.01 ± 2.09
On PND 21	57.33 ± 4.90	56.11 ± 5.54	56.41 ± 6.04	54.16 ± 2.82*

a Values are given as the mean ± S.D.

b Sex ratio = total no. of male pups/total no. of pups.

c Viability index on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) × 100.

d Viability index on PND 4 (%) = (no. of live pups on PND 4/no. of live pups on PND 0) × 100.

e Viability index on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) × 100.

* Signiﬁcantly different from the control, P < 0.05.

** Signiﬁcantly different from the control, P < 0.01.

Table 3

Absolute and relative organ weight of F1 male and female weanlings

AS (ppm)		0 (control)	120	600	3000
Males
No. of animals		22	20	22	22
Body weight	(g)	90.8 ± 6.9	93.4 ± 10.5	89.7 ± 6.1	79.4 ± 7.5^**
Brain	(g)	1.69 ± 0.06	1.73 ± 0.08	1.72 ± 0.07	1.68 ± 0.05
	(g/100g bw)	1.88 ± 0.13	1.87 ± 0.19	1.92 ± 0.09	2.14 ± 0.17^**
Thymus	(mg)	375 ± 55	384 ± 86	357 ± 58	305 ± 51^**
	(mg/100 g bw)	414 ± 56	409 ± 64	398 ± 59	383 ± 36
Liver	(g)	4.33 ± 0.43	4.40 ± 0.60	4.22 ± 0.45	3.49 ± 0.53^**
	(g/100 g bw)	4.77 ± 0.30	4.71 ± 0.33	4.70 ± 0.27	4.37 ± 0.30^**
Kidney^a	(g)	1.06 ± 0.09	1.09 ± 0.14	1.03 ± 0.11	0.95 ± 0.13^**
	(g/100 g bw)	1.17 ± 0.06	1.16 ± 0.07	1.15 ± 0.08	1.20 ± 0.07
Spleen	(mg)	394 ± 49	410 ± 68	388 ± 74	301 ± 43**
	(mg/100 g bw)	436 ± 63	437 ± 40	432 ± 73	379 ± 37**
Testisa	(mg)	596 ± 65	583 ± 67	569 ± 65	539 ± 51*
	(mg/100 g bw)	657 ± 64	626 ± 49	635 ± 64	682 ± 58
Epididymis^a	(mg)	81.8 ± 8.6	76.8 ± 10.9	76.5 ± 8.4	72.0 ± 9.9^**
	(mg/100 g bw)	90.4 ± 10.3	82.0 ± 6.1	85.4 ± 8.4	91.5 ± 14.6
Females
No. of animals		22	20	22	21
Body weight	(g)	84.3 ± 6.3	85.9 ± 9.2	80.5 ± 7.0	75.8 ± 6.4^**
Brain	(g)	1.64 ± 0.06	1.66 ± 0.06	1.63 ± 0.05	1.63 ± 0.07
	(g/100 g bw)	1.96 ± 0.12	1.95 ± 0.18	2.04 ± 0.17	2.16 ± 0.14^**
Thymus	(mg)	383 ± 66	373 ± 74	345 ± 46	313 ± 33^**
	(mg/100 g bw)	453 ± 63	433 ± 64	429 ± 57	415 ± 41
Liver	(g)	3.83 ± 0.47	3.92 ± 0.48	3.61 ± 0.35	3.24 ± 0.34^**
	(g/100 g bw)	4.53 ± 0.30	4.57 ± 0.31	4.48 ± 0.30	4.27 ± 0.25^*
Kidneya	(g)	0.99 ± 0.11	0.99 ± 0.09	0.93 ± 0.10	0.93 ± 0.10
	(g/100 g bw)	1.17 ± 0.08	1.15 ± 0.07	1.15 ± 0.09	1.23 ± 0.09
Spleen	(mg)	337 ± 62	356 ± 55	341 ± 64	292 ± 43^*
	(mg/100 g bw)	400 ± 67	415 ± 44	422 ± 53	386 ± 47
Ovarya	(mg)	25.3 ± 4.8	25.3 ± 3.8	22.5 ± 4.6	24.7 ± 3.2
	(mg/100 g bw)	30.1 ± 5.1	29.7 ± 5.0	27.9 ± 5.0	32.5 ± 4.2
Uterus	(mg)	70.6 ± 16.6	74.2 ± 32.0	59.2 ± 11.9^*	55.4 ± 13.4^**
	(mg/100 g bw)	83.8 ± 19.2	85.5 ± 32.4	73.3 ± 11.9	73.3 ± 18.0

Values are given as the mean ± S.D.

a Values represent the total weights of the organs on both sides.

* Signiﬁcantly different from the control, P < 0.05.

** Signiﬁcantly different from the control, P < 0.01.

Table 4

Absolute and relative organ weight of F2 male and female weanlings

AS (ppm)		0 (control)	120	600	3000
Males
No. of animals		21	18	22	21
Body weight	(g)	87.7 ± 5.8	89.0 ± 8.7	87.0 ± 9.6	79.2 ± 6.8^**
Brain	(g)	1.66 ± 0.05	1.69 ± 0.06	1.70 ± 0.06	1.67 ± 0.06
	(g/100 g bw)	1.90 ± 0.13	1.91 ± 0.17	1.97 ± 0.16	2.13 ± 0.17^**
Thymus	(mg)	382 ± 50	348 ± 49	357 ± 66	305 ± 36^**
	(mg/100 g bw)	439 ± 70	392 ± 52^*	411 ± 57	386 ± 40^**
Liver	(g)	3.93 ± 0.37	4.04 ± 0.64	3.91 ± 0.39	3.45 ± 0.41^**
	(g/100 g bw)	4.49 ± 0.34	4.52 ± 0.44	4.50 ± 0.24	4.36 ± 0.23
Kidney^a	(g)	1.02 ± 0.09	1.01 ± 0.13	0.99 ± 0.13	0.94 ± 0.10
	(g/100 g bw)	1.16 ± 0.08	1.14 ± 0.06	1.14 ± 0.07	1.19 ± 0.06
Spleen	(mg)	368 ± 54	381 ± 62	361 ± 49	296 ± 48^**
	(mg/100 g bw)	421 ± 64	427 ± 50	416 ± 48	372 ± 42^**
Testis^a	(mg)	559 ± 67	549 ± 98	543 ± 77	534 ± 54
	(mg/100 g bw)	637 ± 60	615 ± 81	624 ± 47	680 ± 92
Epididymis^a	(mg)	75.3 ± 6.9	78.3 ± 8.8	75.1 ± 10.7	70.5 ± 5.7^*
	(mg/100 g bw)	86.1 ± 8.3	88.4 ± 9.0	86.5 ± 9.0	89.4 ± 8.2
Females
No. of animals		22	18	21	21
Body weight	(g)	80.8 ± 6.0	80.0 ± 7.2	80.8 ± 9.1	73.8 ± 4.4^**
Brain	(g)	1.60 ± 0.06	1.61 ± 0.05	1.64 ± 0.05*	1.61 ± 0.04
	(g/100 g bw)	1.99 ± 0.14	2.03 ± 0.16	2.05 ± 0.20	2.19 ± 0.15^**
Thymus	(mg)	337 ± 45	364 ± 36	347 ± 49	312 ± 37
	(mg/100 g bw)	419 ± 61	457 ± 50	431 ± 47	424 ± 54
Liver	(g)	3.56 ± 0.35	3.61 ± 0.39	3.61 ± 0.48	3.07 ± 0.26^**
	(g/100 g bw)	4.41 ± 0.21	4.51 ± 0.26	4.47 ± 0.26	4.17 ± 0.29^**
Kidney^a	(g)	0.95 ± 0.07	0.93 ± 0.10	0.92 ± 0.10	0.88 ± 0.08
	(g/100 g bw)	1.18 ± 0.08	1.16 ± 0.09	1.14 ± 0.06	1.20 ± 0.07
Spleen	(mg)	320.9 ± 46.7	331.8 ± 59.3	331.3 ± 57.1	269.9 ± 55.2^**
	(mg/100 g bw)	398.4 ± 59.0	414.8 ± 64.3	409.0 ± 42.2	365.0 ± 67.4
Ovary^a	(mg)	23.9 ± 3.7	22.8 ± 3.6	23.2 ± 3.5	20.2 ± 2.3^**
	(mg/100 g bw)	29.7 ± 4.9	28.8 ± 5.6	29.0 ± 4.7	27.5 ± 3.5
Uterus	(mg)	60.5 ± 17.0	63.8 ± 18.4	65.0 ± 41.7	49.3 ± 11.6^*
	(mg/100 g bw)	74.6 ± 19.2	79.3 ± 19.3	78.7 ± 40.4	67.0 ± 16.2

Values are given as the mean ± S.D.

a Values represent the total weights of the organs on both sides.

* Signiﬁcantly different from the control, P < 0.05.

** Signiﬁcantly different from the control, P < 0.01.

Conclusions:

In a two-generation study rats were continuously exposed to Aluminum sulfate via drinking water. The resulting NOAEL was 600 ppm (41.0 mg/kg bw/day) and the LOAEL was 3000 ppm (188.0 mg/kg bw/day).

Executive summary:

In a 2-generation reproduction study according to OECD 416, Aluminium sulfate (98.5 % purity) was administered to Crl:CD(SD) rats in water at dose levels of 0, 120, 600 and 3000 ppm (0, 8.6, 41.0 and 188 mg/kg bw/day).

No compound related effects were noted in the main categories of systemic or reproductive toxicity evaluated, except a transiently decreased body weight in the 3000 ppm dose group. Moreover, in F1 and F2 pups, the preweaning body weight gain was inhibited at 3000 ppm and the liver and spleen weights were decreased at weaning. At this dose, vaginal opening was slightly delayed. There were no compound-related changes in other reproductive/developmental parameters, including developmental neurobehavioral endpoints.

Based on these findings, the LOAEL is 3000 ppm (188 mg/kg bw/day) and the NOAEL for parental systemic toxicity and reproductive/developmental toxicity is 600 ppm (41 mg/kg bw/day), which equals a daily aluminium intake of 8.06 mg Al/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a 2-generation reproductive study according to OECD 416 in rat.

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