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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Taimei Chemicals Co., Ltd. (Nagano, Japan), Lot No. A81009
- Purity: 99.5%

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test article was dissolved in ion-exchanged water, and served as drinking water for the animals

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) 4 wks; (F1) x wks
- Weight at study initiation: no data
- Housing: individually, except for the acclimation, mating and nursing periods in metal bracket-type cages with wire-mesh floors
- Diet (e.g. ad libitum): standard rat diet: CRF-1; Oroental Yeasr Co., Ltd, Tokyo, Japan, ad libitum
Aluminium concentration in the standard diet, analyzed by flame atomic absorption spectrometry for each lot of diet, ranged from 22 to 29 ppm
- Water (e.g. ad libitum): drinking water containing different concentrations of AAS;
- Acclimation period: 8 days

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

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the test, AAS was dissolved in ion-exchanged water at concentations of 0.05 and 10 mg/mL.
The stability of AAS in ion-exchanged water was confirmed after 5-day storage at room temperature following 7-day refrigerated storage.

DIET PREPARATION
AAS was given in the drinking water. The amount of Aluminium in the fed was analzyed.

VEHICLE
- Concentration in vehicle: 0, 50, 500 and 5000 ppm

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and or sperm in vaginal smear referred to as day 0 of gestation
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female returned to their home cages and allowed to deliver spontaneously

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of AAS in the drinking water was analyzed in the first and last preparation and every 3 month, and were confirmed to be 99.4-104.4% of the target by high performance liquid chromatography.
Concentration of Aluminium in each lot of standard diet was tested by flame atomic absorption spectrometry and ranged from 22 to 29 ppm

Duration of treatment / exposure:

F0: male and female: 10-week administration prior mating, mating and duration of pregnancy of females; males: necropsied at time of pub delivery; females: lactation
F1: designated as F1 parents at postnatal day (PND) 21-25; day of selection was day 0 of a 10-week dosing prior mating; dosing in F1 generation followed than the scheme of F0 generation.
F1 pubs not selected for F1 parents and all F2 weanlings were necropsied on PND 26.

Frequency of treatment:

AAS was given in the drinking water.

Details on study schedule:

The study began with 24 rats/sex/group (F0 generation), and they were exposed to AAS in drinking water at 0, 50, 500 or 5000 ppm. After 10-week administration of AAS, each female was mated with a male from the same dosage group, and pregnant females were allowed to deliver and nurse their pups. F0 parental male rats were necropsied after the parturition of paired females, and F0 females were necropsied after weaning of their pups. Administration of AAS was continued throughout the mating, gestation and lactation periods until necropsy. For the second generation, 24 male and 24 female weanlings (1 or 2 weanlings/sex in each litter) in each group were selected as F1 parents on PNDs 21–25 to equalize the mean body weights among groups as much as possible. The day on which F1 parental animals were selected was designated as day 0 of dosing for the F1 generation. F1-selected rats were given drinking water with the respective formulation, and mated after 10-week administration. They were 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.

No. of animals per sex per dose:

F0 and F1: 24 rats/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale:
Prior dose-finding study in male and female rats (6/sex/dose) given AAS at 0, 300, 1000, 3000 and 10000 ppm. Males were dosed 7-weeks, beginning 14 day before mating, females were dosed from 14 days prior mating to day 4 of lactation throughout the mating and gestation period. AAS reduced water consumption in all treatment groups, and there were decreases in body weight at 3000 ppm and above. At necropsy, thickening of the limiting ridge in the stomach was detected at 10000 ppm, although no animals died at any dose. There were no changes in any other reproductive/developmental parameters.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general appearance and behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: males: 1/week; females: 1/week until evidence of copulation was detected and thereafter of nays 0, 7, 14 and 20 of gesation and on days 0, 4, 7, 14 and 21 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: 1/d
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: 1/week

OTHER: Neurobehavioural evaluation:
Spontanous locomotor activity was evaluated at 4 weeks of age for 10 male and 10 female F1 rats randomly selected from each group.
Spatial learning ability was assessed using a water-filled multiple T-maze (Biel´s type) for 10 male and 10 female F1 rats selected from each group at 6 weeks of age.

Oestrous cyclicity (parental animals):

During the premating period a few AAS-treated F0 and F1 females dad persistent diestrus, however, the incidence of females with normal estrous cycles (4-5 days) was not changed significantly compared with the control.
There were no significant differences in the estrous cycle between control and AAS-treated groups.

Sperm parameters (parental animals):

Parameters examined in F0 and F1 male parental generations:
number of testis sperm and cauda epididymal sperm, percentage of motile sperm and progressively motile sperm, swimming speed and pattern, percetage of morphologically abnormal sperm between control and AAS-treated groups

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, litter size was adjusted to 8 pups/litter (4/sex/litter).

PARAMETERS EXAMINED
The following parameters were examined in F0/F1 offspring (F1 and F2 pubs, respectively):
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight on day 0, 4, 7, 14 and 21,
pinna unfolding daily for 4 days after birth
one male and one female F1 and F2 pub selected from each dam were observed for incisor eruption beginning on PND 8 and eye opening beginning on day 12 until each pup achieved the criterium. For the same F1 and F2 pups, the surface righting relfex, negative geotaxis and mid-air reflex were assessed on PND 5, 8 and 18, respectively.
All F1 offspring selected as F1 parents were observed daily vor male preputial separation beginning on PND 35 of female vaginal opening beginning on PND 25 until completion. The body weight of the respective F1 rats was recorded on the day the criteria were fulfilled.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
F0 and F1 parental males were euthanized by exsanguination under ether anesthesia after the parturition of paired females.
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]
Females were evaluated for estous cycle stage by examination of the vaginal smear after weaning of pups, and euthanized in the proestrus stage by exsanguination under ehter anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The number of uterine implantation sites was recorded in each female.
The tissues indicated in Table [#] were weighed. The testis and epididymis were fixed in Bouin´s solution and preserved in 70% ethanol, and the other organs were stored in 10% neutral-buffered formalin.
Histopathological evaluations were performed for the testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina of all F0 and F1 animals in the control and highest dose groups. These organs were embedded in paraffin by a routine procedure. They were then sectioned stained and hematoxylin-eosin and examined histopathiologically under a light microscope.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at post natal day 26.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
For one male and one female F1 and F2 weanling selected from each dam, major organs were removed, fixed and preserved, as descirbed for the adults.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were and weighed.
In the highest dose group, histopahological evaulations of thymus, liver and spleen were performed in 10 male and 10 female F1 and F2 wanlings. The examined animals were selected randomly from the animals whose organs were stored.

Statistics:

The body weight of parental animals, food and water consumption, length of estrous cycle, gestational length, 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 were evaluated by Bartlett’s test for homogeneity of variances (P < 0.05). The body weight of preweaning pups, AGD, viability and age at the completion of developmental landmarks were similarly analyzed using the litter as the experimental unit. When homogeneity was recognized, one-way analysis of variance was applied (P < 0.10). If a significant difference was found, Dunnett’s test was used for pairwise comparisons between control and individual treatment groups (P < 0.01 or 0.05). Data without homogeneity were subjected to the Kruskal–Wallis rank sum test (P < 0.10), and if significant differences were detected, the Mann–Whitney U test was used to compare AAS-treated groups with the control group (P < 0.01 or 0.05). The incidence of parental animals with clinical signs and necropsy 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 were compared between the control and each dosage group using Fisher’s exact test (P < 0.01 or 0.05). Wilcoxon’s rank sum test was performed for the incidence of pups with clinical signs or necropsy findings per litter, completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex (P < 0.01 or 0.05). The number of primordial follicles was compared between the control and highest dose groups using Student’s t-test (P < 0.01 or 0.05).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

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

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

see below

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

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:

no effects observed

Description (incidence and severity):

see below

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption was significantly lower during week 1 of dosing in F0 males of the 5000 ppm group and in F0 females of the 500 and 5000 ppm groups. In 5000 ppm-treated F0
and F1 females, there were also significant decreases in food consumption in the 2nd and 3rd weeks of lactation.
Body weight was significantly lower in the 2nd week of dosing in both sexes of F0 rats and on day 21 of lactation in F0 females at 5000 ppm. In the 5000 ppm group, the body weight of F1 males and females was significantly lower in the first 2 and 3 weeks of dosing, respectively.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Significant changes were observed throughout or almost throughout the dosing period in F0 males of all AAS-treated groups, and in F0 females and F1 males and females in the 500 and 5000 ppm groups. Significant decreases in water consumption were also found during weeks 1, 9 and 10 of dosing, week 1 of gestation and week 1 of lactation in 50 ppm-treated F0 females and during weeks 4 and 8–10 of dosing in 50 ppm-treated F1 females.
Based on water consumption and body weight, daily AAS 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 AAS-treated groups.

Calculated mean AAS intake during the whole period was:
3.78, 33.5 and 305 mg/kg bw/day in F0 males,
6.52, 58.6 and 500 mg/kg bw/day in F0 females,
4.59, 41.8 and 372 mg/kg bw/day in F1 males, and
6.65, 61.9 and 517 mg/kg bw/day in F1 females,
for the 50, 500 and 5000 ppm groups, respectively.

Considering aluminium content in the basal diet, dietary aluminium exposure of F0 and F1 animals was estimated from the food consumption and body weight in the control and AAS-treated groups. Average aluminium intake from drinking water and food combined was calculated to be:
1.56, 1.98, 5.35 and 36.3 mg Al/kg bw/day in F0 males,
2.20, 2.89, 8.81 and 59.0 mg Al/kg bw/day in F0 females,
1.83, 2.35, 6.57 and 44.2 mg Al/kg bw/day in F1 males, and
2.39, 3.10, 9.36 and 61.1 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, a few AAS-treated F0 and F1 female rats had persistent diestrus; however, the incidence of females with normal estrous cycles (4–5 days) was not changed significantly compared with the control. There were no significant differences in the estrous cycle between control and AAS-treated groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm analysis of schedule-sacrificed F0 and F1 adults revealed no significant differences in the number of testis sperm and cauda epididymal 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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Although some animals failed to copulate, impregnate or deliver live pups, no significant changes were found in the copulation, fertility or gestation index between the control and AAS-treated groups in F0 and F1 generations. There were also no significant differences in the precoital interval and gestation length in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 females in the 500 and 5000 ppm groups and in F1 males and females in the 5000 ppm group, relative kidney weight was increased significantly. A significant decrease in the absolute weight of the pituitary gland was found in F0 females and in F1 males and females at 5000 ppm. In F1 females, there was also a significant decrease in the absolute thymus weight at 5000 ppm. Further, significant decreases were found in the relative weight of the seminal vesicle in 50 ppm-treated F1 males and in the absolute brain weight in 500 ppm-treated F1 females, but no dose-dependency was found in these changes.

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

no effects observed

Description (incidence and severity):

see below

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption was significantly lower during week 1 of dosing in F0 males of the 5000 ppm group and in F0 females of the 500 and 5000 ppm groups. In 5000 ppm-treated F0
and F1 females, there were also significant decreases in food consumption in the 2nd and 3rd weeks of lactation.
Body weight was significantly lower in the 2nd week of dosing in both sexes of F0 rats and on day 21 of lactation in F0 females at 5000 ppm. In the 5000 ppm group, the body weight of F1 males and females was significantly lower in the first 2 and 3 weeks of dosing, respectively.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Significant changes were observed throughout or almost throughout the dosing period in F0 males of all AAS-treated groups, and in F0 females and F1 males and females in the 500 and 5000 ppm groups. Significant decreases in water consumption were also found during weeks 1, 9 and 10 of dosing, week 1 of gestation and week 1 of lactation in 50 ppm-treated F0 females and during weeks 4 and 8–10 of dosing in 50 ppm-treated F1 females.
Based on water consumption and body weight, daily AAS 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 AAS-treated groups.

Calculated mean AAS intake during the whole period was:
3.78, 33.5 and 305 mg/kg bw/day in F0 males,
6.52, 58.6 and 500 mg/kg bw/day in F0 females,
4.59, 41.8 and 372 mg/kg bw/day in F1 males, and
6.65, 61.9 and 517 mg/kg bw/day in F1 females,
for the 50, 500 and 5000 ppm groups, respectively.

Considering aluminium content in the basal diet, dietary aluminium exposure of F0 and F1 animals was estimated from the food consumption and body weight in the control and AAS-treated groups. Average aluminium intake from drinking water and food combined was calculated to be:
1.56, 1.98, 5.35 and 36.3 mg Al/kg bw/day in F0 males,
2.20, 2.89, 8.81 and 59.0 mg Al/kg bw/day in F0 females,
1.83, 2.35, 6.57 and 44.2 mg Al/kg bw/day in F1 males, and
2.39, 3.10, 9.36 and 61.1 mg Al/kg bw/day in F1 females
for control through high-dose groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the premating period, a few AAS-treated F0 and F1 female rats had persistent diestrus; however, the incidence of females with normal estrous cycles (4–5 days) was not changed significantly compared with the control. There were no significant differences in the estrous cycle between control and AAS-treated groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm analysis of schedule-sacrificed F0 and F1 adults revealed no significant differences in the number of testis sperm and cauda epididymal 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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Although some animals failed to copulate, impregnate or deliver live pups, no significant changes were found in the copulation, fertility or gestation index between the control and AAS-treated groups in F0 and F1 generations. There were also no significant differences in the precoital interval and gestation length in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In F0 females in the 500 and 5000 ppm groups and in F1 males and females in the 5000 ppm group, relative kidney weight was increased significantly. A significant decrease in the absolute weight of the pituitary gland was found in F0 females and in F1 males and females at 5000 ppm. In F1 females, there was also a significant decrease in the absolute thymus weight at 5000 ppm. Further, significant decreases were found in the relative weight of the seminal vesicle in 50 ppm-treated F1 males and in the absolute brain weight in 500 ppm-treated F1 females, but no dose-dependency was found in these changes.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Anogenital distance (AGD):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No significant changes were found in the number of implantations or pups delivered, delivery index, sex ratio of pups and the viability index during the preweaning period in either generation.

BODY WEIGHT (OFFSPRING)
While there were no significant differences in birth weight between the control and AAS-treated groups, the body weight of F1 males on PND 21 and ofF1 females on PNDs 14 and 21 was significantly lower in the 5000 ppm group than in the control. A similar decreasing trend was found in the body weight of male and female F2 pups around the time of weaning in the highest dose group, although no statistical significance was
found.

SEXUAL MATURATION (OFFSPRING)
In F1 female animals, vaginal opening was significantly delayed at 5000 ppm (32.3 ± 1.8 days of age, compared with 30.2 ± 2.1 days of age in controls, P 6 0.01). Body weight at the time of attainment was not significantly, but was slightly heavier in this 5000 ppm group (122.0 ± 15.7 g, compared with 115.8 ± 12.6 g in control). There were no significant differences in age at preputial separation or body weight at the time of completion in F1 males between control and AAS-treated groups.

ORGAN WEIGHTS (OFFSPRING)
In F1 and F2 weanlings, 5000 ppm-treated males and females had significantly lower body weights, and the absolute and relative weights of the spleen in both sexes and of the thymus in males were significantly decreased in this 5000 ppm group. A decrease in the absolute thymus weight was also observed in F1 females given 500 and 5000 ppm and in F2 females given 5000 ppm, but there were no significant changes in relative weight in F1 or F2 females. The absolute liver weight was significantly decreased in F1 and F2 males and females, accompanied with a decrease in the relative weight in F1 males and F2 females in the 5000 ppm group. The relative weights of the brain and kidney were increased significantly in F1 and F2 males and females given 5000 ppm. Further, a significant decrease in the absolute weight of the kidney, adrenal, testis, epididymis, ovary and uterus was found at 500 and/or 5000 ppm.

GROSS PATHOLOGY (OFFSPRING)
During the preweaning period, external gross examination 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; however, there were no significant differences in incidence between the control and AAS treated groups.
No gross abnormalities were found in any F2 pups.

HISTOPATHOLOGY (OFFSPRING)
No dose-related changes were found in the histopathology of the liver and spleen in both
sexes and of the thymus in males in either generation.

OTHER FINDINGS (OFFSPRING)
In F1 and F2 pups, there were no significant differences in the completion rate of pinna unfolding, the age at completion of incisor eruption and eye opening, and AGD and AGD per cube root of the body weight ratio between the control and AAS-treated groups. All male and female F1 and F2 pups in all groups achieved the surface righting reflex on PND 5, negative geotaxis reflex on PND 8 and mid-air righting reflex on PND 18, and no significant changes were found in the response time of surface righting
and negative geotaxis reflex.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Anogenital distance (AGD):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No significant changes were found in the number of implantations or pups delivered, delivery index, sex ratio of pups and the viability index during the preweaning period in either generation.

BODY WEIGHT (OFFSPRING)
While there were no significant differences in birth weight between the control and AAS-treated groups, the body weight of F1 males on PND 21 and ofF1 females on PNDs 14 and 21 was significantly lower in the 5000 ppm group than in the control. A similar decreasing trend was found in the body weight of male and female F2 pups around the time of weaning in the highest dose group, although no statistical significance was
found.

SEXUAL MATURATION (OFFSPRING)
In F1 female animals, vaginal opening was significantly delayed at 5000 ppm (32.3 ± 1.8 days of age, compared with 30.2 ± 2.1 days of age in controls, P 6 0.01). Body weight at the time of attainment was not significantly, but was slightly heavier in this 5000 ppm group (122.0 ± 15.7 g, compared with 115.8 ± 12.6 g in control). There were no significant differences in age at preputial separation or body weight at the time of completion in F1 males between control and AAS-treated groups.

ORGAN WEIGHTS (OFFSPRING)
In F1 and F2 weanlings, 5000 ppm-treated males and females had significantly lower body weights, and the absolute and relative weights of the spleen in both sexes and of the thymus in males were significantly decreased in this 5000 ppm group. A decrease in the absolute thymus weight was also observed in F1 females given 500 and 5000 ppm and in F2 females given 5000 ppm, but there were no significant changes in relative weight in F1 or F2 females. The absolute liver weight was significantly decreased in F1 and F2 males and females, accompanied with a decrease in the relative weight in F1 males and F2 females in the 5000 ppm group. The relative weights of the brain and kidney were increased significantly in F1 and F2 males and females given 5000 ppm. Further, a significant decrease in the absolute weight of the kidney, adrenal, testis, epididymis, ovary and uterus was found at 500 and/or 5000 ppm.

GROSS PATHOLOGY (OFFSPRING)
During the preweaning period, external gross examination 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; however, there were no significant differences in incidence between the control and AAS treated groups.
No gross abnormalities were found in any F2 pups.

HISTOPATHOLOGY (OFFSPRING)
No dose-related changes were found in the histopathology of the liver and spleen in both
sexes and of the thymus in males in either generation.

OTHER FINDINGS (OFFSPRING)
In F1 and F2 pups, there were no significant differences in the completion rate of pinna unfolding, the age at completion of incisor eruption and eye opening, and AGD and AGD per cube root of the body weight ratio between the control and AAS-treated groups. All male and female F1 and F2 pups in all groups achieved the surface righting reflex on PND 5, negative geotaxis reflex on PND 8 and mid-air righting reflex on PND 18, and no significant changes were found in the response time of surface righting
and negative geotaxis reflex.

Table 1 Reproductive performance of F0 and F1 parental animals.
AAS (ppm)		0 (control)	50	500	5000
F0 generation
No. of rats (male/female)		24/24	24/24	24/24	24/24
Copulation index (%)^a	males	100	95.8	91.7	100
	females	100	100	100	100
Precoital inverval (days)^b		2.2±1.0	2.5±1.6	2.3±1.2	2.8±1.6
Fertility index (%)^c	males	100	91.3	100	100
	females	100	87.5	100	100
Gestation index (%)^d		100	100	100	100
Gestation length (days)^b		22.3±0.5	22.4±0.6	22.3±0.5	22.4±0.5

F1 generation
No. of rats (male/female)		24/24	24/24	23/24	24/24
Copulation index (%)^a	males	91.7	91.7	91.3	95.8
	females	100	95.8	100	100
Precoital inverval (days)^b		2.7±1.8	3.0±2.1	3.3±2.4	3.1±1.3
Fertility index (%)^c	males	90.9	77.3	95.2	100
	females	91.7	78.3	95.8	95.8
Gestation index (%)^d		100	100	95.7	100
Gestation length (days)^b		22.3±0.5	22.3±0.5	22.2±0.4	22.2±0.4
^aCopulation index (%) = (No. of animals with successful copulation/No. of animals paired) x 100.
^bValues are given as the mean ± S.D.
^cFertility index (%) = (No. of animals that impregnated a female or were pregnant/No. of animals with successful copulation) x 100.
^dGestation index (%) = (No. of females that delivered live pups/No. of pregnant females) x 100.

Table 2
Developmental findings for F1 and F2 offsprings.
AAS (ppm)	0 (control)	50	500	5000
F0 parents/F1 offspring
No. of F0 pregnant females	24	21	24	24
No. of implantations^a	14.7 ± 3.1	14.3 ± 2.1	15.0 ± 3.3	15.1 ± 1.5
No. of litters	24	21	24	24
No. of pups delivered^a	13.6 ± 3.1	13.5 ± 2.5	13.8 ± 3.1	14.4 ± 1.6
Delivery index (%)^a,b	92.4 ± 8.0	94.2 ± 10.3	92.3 ± 7.8	95.4 ± 5.4
Sex ratio of pups^c	0.509	0.493	0.476	0.487

Viability index of pups (%)^a
On PND 0^d	99.5 ± 2.7	99.0 ± 2.4	99.5 ± 1.7	99.2 ± 2.3
On PND 4^e	98.3 ± 5.0	98.0 ± 5.4	95.6 ± 20.4	99.2 ± 2.3
On PND 21^f	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0

Male pup weight during lactation (g)^a
On PND 0	6.93 ± 0.66	6.96 ± 0.68	6.91 ± 0.48	6.90 ± 0.69
On PND 4	11.13 ± 1.88	10.84 ± 1.47	10.72 ± 0.94	10.68 ± 1.33
On PND 7	19.14 ± 2.30	18.86 ± 2.30	18.71 ± 1.51	18.49 ± 1.70
On PND 14	38.45 ± 3.57	38.32 ± 3.96	37.88 ± 2.31	36.51 ± 2.20
On PND 21	63.83 ± 5.93	62.59 ± 7.09	61.71 ± 4.94	58.67 ± 3.91**

Female pup weight during lactation (g)^a
On PND 0	6.66 ± 0.82	6.57 ± 0.61	6.58 ± 0.57	6.43 ± 0.63
On PND 4	10.70 ± 2.02	10.34 ± 1.25	10.22 ± 1.13	10.13 ± 1.28
On PND 7	18.40 ± 2.49	17.96 ± 2.02	17.97 ± 1.74	17.38 ± 1.79
On PND 14	37.23 ± 3.65	36.97 ± 3.30	36.59 ± 2.74	35.07 ± 2.35*
On PND 21	61.65 ± 6.05	60.03 ± 5.55	59.34 ± 5.22	56.13 ± 4.07**

F1 parents/F2 offspring
No. of F1 parent females	22	18	23	23
No. of implantations^a	15.0 ± 1.6	14.7 ± 1.7	14.7 ± 3.5	14.1 ± 2.2
No. of litters	22	18	22	23
No. of pups delivered^a	13.9 ± 1.8	13.7 ± 2.4	14.0 ± 3.8	13.5 ± 2.1
Delivery index (%)^a,b	92.7 ± 9.4	93.0 ± 11.2	90.9 ± 20.4	95.6 ± 5.7
Sex ratio of pups^c	0.435	0.500	0.492	0.506

Viability index of pups (%)^a
On PND 0^d	98.3 ± 4.5	97.6 ± 4.2	98.9 ± 3.3	99.7 ± 1.5
On PND 4^e	97.9 ± 6.0	99.4 ± 2.4	99.4 ± 1.9	99.0 ± 2.9
On PND 21^f	99.4 ± 2.7	100.0 ± 0.0	100.0 ± 0.0	100.0 ± 0.0

Male pup weight during lactation (g)^a
On PND 0	6.97 ± 0.62	7.03 ± 0.65	6.89 ± 0.53	6.97 ± 0.75
On PND 4	10.64 ± 1.62	11.31 ± 1.22	10.95 ± 1.32	11.16 ± 1.87
On PND 7	17.97 ± 2.18	19.19 ± 1.73	18.82 ± 1.90	18.42 ± 2.39
On PND 14	36.89 ± 3.26	38.99 ± 3.14	38.28 ± 3.26	36.40 ± 3.67
On PND 21	61.07 ± 6.06	64.40 ± 5.58	63.20 ± 5.51	58.65 ± 5.90

Female pup weight during lactation (g)^a
On PND 0	6.46 ± 0.47	6.68 ± 0.67	6.54 ± 0.50	6.51 ± 0.63
On PND 4	9.91 ± 1.26	10.62 ± 1.18	10.30 ± 1.20	10.59 ± 1.72
On PND 7	17.15 ± 2.06	18.28 ± 1.77	17.73 ± 1.68	17.58 ± 2.34
On PND 14	35.58 ± 3.00	37.47 ± 2.74	36.65 ± 2.69	35.20 ± 3.44
On PND 21	58.47 ± 5.33	61.83 ± 4.40	60.05 ± 3.82	56.72 ± 5.39

* Significantly different from the control, P < 0.05.

** Significantly different from the control, P < 0.01

a Values are given as the mean ± S.D.

b Delivery index (%) = (No. of pups delivered/No. of implantations) x 100.

c Sex ratio = total No. of male pups/total No. of pups.

d Viability index on PND 0 (%) = (No. of live pups on PND 0/No. of pups delivered) x 100.

e Viability index on PND 4 (%) = (No. of live pups on PND 4/No. of live pups on PND 0) x 100.

f Viability index on PND 21 (%) = (No. of live pups on PND 21/No. of live pups on PND 4 after cull) x 100.

Table 3
Absolute and relative organ weight of F1 male and female weanlings.
AAS (ppm)		0 (control)	50	500	5000
Males
No. of animals		24	20	23	24
Body weight	(g)	94.1 ± 9.1	90.8 ± 10.7	91.3 ± 9.8	80.9 ± 7.5**
Brain	(g)	1.72 ± 0.08	1.71 ± 0.07	1.70 ± 0.06	1.68 ± 0.07
	(g/100 g b.w.)	1.84 ± 0.16	1.90 ± 0.17	1.88 ± 0.16	2.09 ± 0.15**
Thymus	(mg)	392 ± 67	373 ± 72	360 ± 57	301 ± 48**
	(mg/100 g b.w.)	417 ± 61	411 ± 55	396 ± 61	372 ± 52*
Liver	(g)	4.32 ± 0.54	4.15 ± 0.55	4.12 ± 0.53	3.52 ± 0.43**
	(g/100 g b.w.)	4.58 ± 0.29	4.57 ± 0.17	4.51 ± 0.27	4.34 ± 0.25**
Kidney^a	(g)	1.08 ± 0.13	1.04 ± 0.14	1.05 ± 0.10	0.98 ± 0.10*
	(g/100 g b.w.)	1.15 ± 0.10	1.15 ± 0.08	1.15 ± 0.06	1.21 ± 0.08*
Spleen	(mg)	421 ± 75	399 ± 66	403 ± 91	292 ± 49**
	(mg/100 g b.w.)	447 ± 64	441 ± 60	439 ± 78	361 ± 43**
Adrenal^a	(mg)	26.4 ± 3.4	24.5 ± 2.7	25.5 ± 3.2	24.0 ± 3.4*
	(mg/100 g b.w.)	28.2 ± 3.6	27.2 ± 2.9	28.0 ± 3.0	29.8 ± 3.5
Testis^a	(mg)	591 ± 69	571 ± 74	573 ± 72	532 ± 78*
	(mg/100 g b.w.)	628 ± 38	630 ± 41	628 ± 49	656 ± 61
Epididymis^a	(mg)	80.7 ± 9.3	76.2 ± 10.7	78.9 ± 10.0	67.8 ± 9.9**
	(mg/100 g b.w.)	86.0 ± 8.1	84.3 ± 10.4	86.6 ± 8.3	84.2 ± 11.6

Females
No. of animals		24	21	23	24
Body weight	(g)	87.0 ± 7.2	85.5 ± 7.6	83.3 ± 7.1	76.2 ± 7.0**
Brain	(g)	1.68 ± 0.12	1.64 ± 0.06	1.65 ± 0.06	1.62 ± 0.06
	(g/100 g b.w.)	1.93 ± 0.16	1.93 ± 0.16	1.99 ± 0.14	2.14 ± 0.16**
Thymus	(mg)	382 ± 58	365 ± 48	342 ± 51*	316 ± 41**
	(mg/100 g b.w.)	437 ± 46	429 ± 56	411 ± 54	416 ± 54
Liver	(g)	3.79 ± 0.38	3.80 ± 0.39	3.73 ± 0.42	3.28 ± 0.43**
	(g/100 g b.w.)	4.36 ± 0.38	4.45 ± 0.28	4.48 ± 0.31	4.30 ± 0.28
Kidney^a	(g)	0.98 ± 0.10	0.97 ± 0.11	0.96 ± 0.09	0.93 ± 0.08
	(g/100 g b.w.)	1.13 ± 0.08	1.14 ± 0.06	1.15 ± 0.05	1.22 ± 0.07**
Spleen	(mg)	362 ± 63	351 ± 44	356 ± 59	272 ± 47**
	(mg/100 g b.w.)	416 ± 72	412 ± 49	428 ± 63	356 ± 46**
Adrenal^a	(mg)	25.5 ± 3.9	23.6 ± 3.0	22.7 ± 3.0**	22.3 ± 2.6**
	(mg/100 g b.w.)	29.4 ± 4.0	27.8 ± 3.8	27.3 ± 3.5	29.4 ± 3.1
Ovary^a	(mg)	24.6 ± 4.5	24.6 ± 4.4	23.8 ± 2.9	22.0 ± 4.0
	(mg/100 g b.w.)	28.2 ± 4.6	29.0 ± 4.6	28.9 ± 4.6	29.2 ± 6.4
Uterus	(mg)	67.3 ± 15.3	66.4 ± 21.4	64.6 ± 15.9	50.4 ± 10.9**
	(mg/100 g b.w.)	77.3 ± 16.6	77.1 ± 20.5	77.2 ± 15.8	66.2 ± 12.8

Values are given as the mean ± S.D.

* Significantly different from the control, P < 0.05.

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

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

Table 4
Absolute and relative organ weight of F2 male and female weanlings.
AAS (ppm)		0 (control)	50	500	5000
Males
No. of animals		22	18	22	23
Body weight	(g)	89.9 ± 7.5	94.1 ± 8.3	91.7 ± 7.9	82.9 ± 10.2*
Brain	(g)	1.70 ± 0.06	1.73 ± 0.06	1.70 ± 0.06	1.67 ± 0.08
	(g/100 g b.w.)	1.90 ± 0.17	1.85 ± 0.14	1.86 ± 0.12	2.04 ± 0.23*
Thymus	(mg)	375 ± 69	379 ± 50	365 ± 50	296 ± 53**
	(mg/100 g b.w.)	417 ± 65	404 ± 53	399 ± 46	359 ± 58**
Liver	(g)	4.12 ± 0.55	4.49 ± 0.53	4.34 ± 0.44	3.69 ± 0.48*
	(g/100 g b.w.)	4.57 ± 0.32	4.77 ± 0.34	4.73 ± 0.19	4.46 ± 0.20
Kidney^a	(g)	1.02 ± 0.10	1.08 ± 0.10	1.03 ± 0.10	1.01 ± 0.13
	(g/100 g b.w.)	1.13 ± 0.08	1.15 ± 0.08	1.13 ± 0.07	1.22 ± 0.07**
Spleen	(mg)	390 ± 86	387 ± 48	393 ± 40	292 ± 52**
	(mg/100 g b.w.)	435 ± 93	413 ± 54	430 ± 41	352 ± 46**
Adrenal^a	(mg)	26.0 ± 3.8	25.2 ± 3.5	25.5 ± 3.3	24.7 ± 4.3
	(mg/100 g b.w.)	29.0 ± 4.1	26.7 ± 3.3	28.0 ± 3.9	29.8 ± 3.0
Testis^a	(mg)	546 ± 83	571 ± 83	572 ± 70	515 ± 67
	(mg/100 g b.w.)	607 ± 72	605 ± 59	623 ± 51	624 ± 65
Epididymis^a	(mg)	74.8 ± 7.5	76.1 ± 10.6	75.1 ± 9.9	68.7 ± 9.1
	(mg/100 g b.w.)	83.7 ± 10.2	80.7 ± 7.5	82.0 ± 9.0	83.6 ± 11.5

Females
No. of animals		22	18	22	23
Body weight	(g)	85.3 ± 7.2	87.6 ± 6.5	84.0 ± 4.9	77.2 ± 5.7**
Brain	(g)	1.65 ± 0.05	1.65 ± 0.06	1.64 ± 0.06	1.62 ± 0.06
	(% of body weight)	1.95 ± 0.16	1.89 ± 0.13	1.95 ± 0.08	2.11 ± 0.15**
Thymus	(mg)	367 ± 68	354 ± 60	352 ± 47	300 ± 40**
	(mg/100 g b.w.)	432 ± 84	405 ± 66	419 ± 48	390 ± 55
Liver	(g)	3.93 ± 0.41	3.97 ± 0.37	3.80 ± 0.33	3.33 ± 0.34**
	(g/100 g b.w.)	4.61 ± 0.25	4.54 ± 0.19	4.52 ± 0.25	4.31 ± 0.31**
Kidney^a	(g)	0.96 ± 0.08	0.97 ± 0.09	0.94 ± 0.06	0.93 ± 0.08
	(g/100 g b.w.)	1.13 ± 0.07	1.11 ± 0.07	1.12 ± 0.05	1.21 ± 0.07**
Spleen	(mg)	355 ± 53	330 ± 33	349 ± 52	276 ± 35**
	(mg/100 g b.w.)	416 ± 51	378 ± 35*	415 ± 59	358 ± 42**
Adrenal^a	(mg)	23.3 ± 2.3	23.2 ± 2.3	23.2 ± 3.4	23.2 ± 2.4
	(mg/100 g b.w.)	27.4 ± 2.7	26.6 ± 2.9	27.5 ± 3.5	30.0 ± 3.1*
Ovary^a	(mg)	24.6 ± 3.0	24.9 ± 4.0	24.2 ± 4.1	20.4 ± 3.2**
	(mg/100 g b.w.)	29.1 ± 4.3	28.5 ± 4.0	29.0 ± 5.5	26.7 ± 4.9
Uterus	(mg)	71.0 ± 55.7	66.8 ± 16.5	58.5 ± 11.8	53.5 ± 11.1*
	(mg/100 g b.w.)	82.3 ± 59.4	76.0 ± 15.4	69.6 ± 12.4	69.5 ± 14.8

Values are given as the mean ± S.D.

* Significantly different from the control, P < 0.05.

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

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

Conclusions:

In a two-generation study continuously exposing rats to aluminum ammonium sulfate via drinking water resulted in a NOAEL level of 500 ppm, which equals a dose of 5.35 mg Aluminum/kg bw/day. The LOAEL was 5000 ppm in this study.

Executive summary:

In a two-generation reproduction study (OECD 416) aluminium ammonium sulfate (99.5 % purity) was continuously given to groups of Crl:CD(SD) rats via drinking water at levels of 0, 50, 500 and 5000 ppm. Male and female rats of F0 and F1 received AAS from 10-weeks prior mating, mating and for the duration of female´s pregnancy. Males were sacrificed after parturition and females further received AAS during lactation. On PNDs 21-25 animals were selected as F1 parents marking day 0 of the 10-week prior mating phase.

Water consumption was decreased in all AAS-treated groups, and the body weight of parental animals transiently decreased in the 5000 ppm group. In either generation, no compound-related changes were found in estrous cyclicity, sperm parameters, copulation, fertility and gestation index, number of implantations and live birth pups, sex ratios of pups or viability during the preweaning period.

Male and female F1 pups in the 5000 ppm group showed a lower body weight on postnatal day 21, while there were no differences in the birth weight of F1 and F2 pups between the control and AAS-treated groups. Preweaning body weight gain in F2 males and females indicated a similar decreasing tendency at 5000 ppm. In F1 and F2 weanlings, the weight of the liver, spleen and thymus decreased at 5000 ppm, but no histopathological changes were found in these organs. In F1 females in the 5000 ppm group, vaginal opening was delayed slightly. There were no compound-related changes in male preputial separation or in other developmental landmarks. In behavioral tests conducted for F1 animals at 4–6 weeks of age, no compound-related changes were found in spontaneous locomotor activity and performance in a water-filled multiple T-maze. In conclusion, the NOAEL of AAS for two-generation reproductive/developmental toxicity was considered to be 500 ppm in rats, primarily based on the depression of preweaning body weight gain. Considering the aluminium content in the basal diet, the total ingested dose of aluminium from drinking water and food in this 500 ppm group was calculated to be 5.35 mg Al/kg bw/day.

The two-generation study in the rat is classified acceptable and satisfies the guideline requirements for a development toxicity study (OECD 416) in rat.

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ALFERROCK

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