Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2008 - 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (Spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured).

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured
Qualifier:
equivalent or similar to guideline
Guideline:
other: Japanese guidelines for the "designation of food additives and for the revision of standards for the use of food additives".
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium sulphate
EC Number:
233-135-0
EC Name:
Aluminium sulphate
Cas Number:
10043-01-3
Molecular formula:
Al.3/2H2O4S
IUPAC Name:
Aluminium sulphate
Details on test material:
- Name of test material (as cited in study report): aluminium sulfate (anhydrous) - AS
- Analytical purity: 98.5 %
- Lot/batch No.: 007x1828
- Stability under test conditions: the test item was stable in ion-exchanged water at concentrations of 0.1, 0.6 and 15 mg/L at least for 10 days (4 days storage at room temperature following 6 days refrigerated storage); dosing solutions were prepared at least every 6 days and kept under cool conditions until serving.
- Storage condition of test material: kept in a sealed container under cool and dark conditions.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: (P) 5 wks; (F1) 3 wks
- Housing: animals were housed individually (except during the acclimation, mating and nursing periods) in suspended wire-mesh cages. From day 17 of gestation to day 21 after delivery, the wire-mesh floor of the cage was replaced with a stainless-steel tray, and individual dams and litters were reared using wood chips as bedding.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet: standard rat diet (CRF-1;Oriental YeastCo.,Ltd.,Tokyo,Japan), ad libitum
- Water: ion-exchanged drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 36-59
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

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

Examinations

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

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

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

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

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

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

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

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

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

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

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

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

HISTOPATHOLOGY / ORGAN WEIGTHS
For one male and one female F1 and F2 weanlings selected from each dam:
- the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries were removed and prepared for microscopic examination and weighed
- Since test substance-related organ weight changes were found in liver and spleen of highest dose group in F1 and F2 generations, these tissues were histopathologically examined for 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups
- If treatment-related histopathological changes were observed in the highest dose group, the same tissue in the next lower dose group was examined as well.
Statistics:
Bartlett's test: was applied for homogeneity of distribution for parametric data (body weight, food and water consumption, length of estrous cycle and gestation, precoital interval, the number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, behavioural test parameters, organ weight and sperm parameters);
For preweaning pups, body weight, AGD, viability, and age at the completion of developmental landmarks were similarly analyzed using the litter as the experimental unit.
One way analysis of variance was performed when the homogeneity of distribution was established.
If a significant difference was detected, Dunnett’s test was conducted for comparison between control and individual treatment groups.
Data without homogeneity were analyzed using the Kruskal-Wallis rank sum test. If significant differences were found, the Mann Whitneys’s U test was conducted for comparison between the control and each dose group.
Fisher exact test was used to compare the incidence of parental animals with clinical signs, and autopsy and histopathological findings, the incidence of females with normal estrous cycle, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ration and completion rate of negative geotaxis between the AS and control group.
The Wilcoxon rank sum test was used to analyze the incidence of pups with clinical signs and necroscopy findings per litter, the completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex.
Student's T-test was used to compare the number of primordial follicles in the control and highest dose group because the homogeneity of variance was indicated by the F-test.
All these statistical analysis were conducted using the 5% level of probability as the criterion for significance.
Reproductive indices:
- Copulation index (for males and females) (%): (no. of animals with successful copulation/no. of animals paired) x 100
- precoital interval (days)
- fertility index (for males and females) (%): (no. of males that impregnated a female or no. of pregnant/no. of animals with successful copulation) x 100
- Gestation index (%): (no. of females that delivered live pups/no. of pregnant females) x 100
- Gestation length (days)
- Delivery index (%): (no. of pups delivered/no. of implantations) x 100
- Estrous cycle in P0 and F1 females
Offspring viability indices:
For F1 and F2 offspring:
Maternal indices; no. of litters; no. of pups delivered; sex of all pups; sex ration of pups total (no. of male pups/total no. of pups)
Viability index calculated:
on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) x 100
on PND 4 (%) = (no. of live pups on PND 4/ no. of live pups on PND 0) x 100
on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no significant difference seen between control and AS treated groups in incidence of clinical signs; 120 ppm: one F1 female died (non adverse); 600 ppm: one P female died (non adverse); 3000 ppm: one F1 female died (non adverse)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: administered in drinking water

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant difference was seen between control and AS treated groups in the incidence of clinical signs of toxicity in either malel or female P and F1 rats.
120ppm: One F1 male died at 9 weeks of dosing. Soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. Necropsy revealed accumulation of ascitic and pleural fluid and dark purple discoloration of liver and kidneys
600ppm: One P female died at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this female from the beginning of 5 weeks of dosing
3000ppm: One F1 male died at 12 weeks of dosing. No clinical signs of toxicity were observed.

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

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

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

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

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

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

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

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


Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
31.2 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: Decreased body weight gain, decreased food consumption
Dose descriptor:
NOAEL
Effect level:
8.06 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Dose descriptor:
LOAEL
Effect level:
31.2 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: Decreased preweaning body weight gain in the F1 and F2 males and females; decreased absolute and relative liver and spleen weights in he F1 and F2 males and females; delayed vaginal opening in F1 females
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Dose descriptor:
NOAEL
Effect level:
8.06 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
2 F1 pups showed malformations (non adverse)
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: F1 males and females - body weights were significantly lower on PND21; F2 females - body weights were significantly lower than controls on PND21
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: vaginal opening was significantly delayed (F1)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: absolute and relative liver weights and absolute spleen weight, absolute weight of thymus, kidney, testes and epididymides decreased; absolute weight of uterus decreased at 600 ppm; relative brain weight decreased
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
F1 and F2 generation - 120, 600 and 3000 ppm:
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation.

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

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

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

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

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

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

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

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

BEHAVIOR PERFORMANCE
F1 males and females - 120, 600 and 3000 ppm
Spontaneous locomotor activity was not significantly different between control and AS- treated groups at 10-min intervals and for 60 min.
Learning and memory performance in T-maze test
Pre-test swimming trials in the straight channel
- no differences between male and female rats in each group compared to the controls;
- no significant changes in the elapsed time to traverse the straight channel;
- in males, no significant changes in the elapsed time and number of errors on days 2–4;
- in females, the elapsed time and the number of errors was significantly lowered at 600 ppm on day 2, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 between control and AS-treated groups (data not shown).

Effect levels (F1)

open allclose all
Dose descriptor:
LOAEL
Generation:
other: F1 and F2
Effect level:
31.2 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
other: Decreased preweaning body weight gain in the F1 and F2 males and females; decreased absolute and relative liver and spleen weights in he F1 and F2 males and females; delayed vaginal opening in F1 females
Dose descriptor:
NOAEL
Generation:
other: F1 and F2
Effect level:
8.06 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 2: Reproductive performance of P0 and F1 parental animals

 AS (ppm)

 

 0

 120

 600

 3000

 P generation

 

 

 

 

 

 No. of rats (male/female)

 

 24/24

 24/24

 24/24

 24/24

 Copulation index (%)

 males

 91.7

 91.7

 100

 91.7

 

 females

 95.8

 100

 100

 100

 Precoital interval (days)

 

 3.2 ± 1.1

 3.2 ± 1.8

 2.9 ± 1.3

 2.8 ± 1.6

 Fertility index (%)

 males

 95.5

 90.9

 100

 95.5

 

 females

 95.7

 91.7

 100

 95.8

 Gestation index (%)

 

 100

 95.5

 95.7

 95.7

 Gestation length (days)

 

 22.4 ± 0.5

 22.5 ± 0.6

 22.1 ± 0.4

 22.3 ± 0.5

 Delivery index (%)

 

 94.3 ± 5.6

 88.6 ± 21.0

 90.7 ± 20.8

 92.0 ± 20.5

 F1 generation

 

 

 

 

 

 No. of rats (male/female)

 

 24/24

 23/24

 24/24

 24/24

 Copulation index (%)

 males

 95.8

 91.3

 95.8

 87.5

 

 females

 100

 95.8

 100

 95.8

 Precoital interval (days)

 

 3.3 ± 3.2

 3.0 ± 2.0

 2.7 ± 1.5

 2.3 ± 1.1

 Fertility index (%)

 males

 91.3

 81.0

 91.3

 95.2

 

 females

 91.7

 82.6

 91.7

 91.3

 Gestation index (%)

 

 100

 94.7

 100

 100

 Gestation length (days)

 

 22.4 ± 0.5

 22.3 ± 0.5

 22.2 ± 0.4

 22.2 ± 0.4

 Delivery index (%)

 

 94.0 ± 9.9

 87.5 ± 22.6

 91.4 ± 10.7

 94.6 ± 6.8

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

 As (ppm)

 0   

 120   

 600   

 3000   

 Organ weight

 F1 males

 F2 males

 F1 males

 F2 males

 F1 males

F2 males

 F1 males

 F2 males

 number of animals

 22

 21

 20

 18

 22

 22

 22

 21

 body weight (g)

 100%

 100%

 NS

 NS

 NS

 

 87.44**

 90.31**

 brain                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

relative weight (g/100g bw) 

 100%

 100%

 NS

 NS

 NS

 NS

 113.22**

 112.11**

 thymus                        

 absolute weight (g)

 100%

 100%

 NS

 

 NS

 NS

 81.33**

 79.84**

 relative weight (g/100g bw)

 100%

 100%

 NS

 89.29*

 NS

 NS

 NS

 87.92**

 Livera                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 80.60**

 87.78**

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 91.61**

 NS

 Kidneya                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 89.62**

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 spleen                         

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 76.40**

 80 .43

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 86.93**

 88.36**

 testisa                        

absolute weight (g) 

 100%

 100%

 NS

 NS

 NS

 NS

 90.44*

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 Epididymisa                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 88.02**

 93.62*

relative weight (g/100g bw) 

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

NS- not statistically significant compared to untreated control

**- significantly different from control, p<0.05

*- significantly different from control, p< 0.01

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

 As (ppm)

 0   

 120   

 600   

 3000   

 Organ weight

 F1 females

 F2 females

 F1 females

 F2 females

 F1 females

F2 females

 F1 females

 F2 females

 number of animals

 22

 22

 20

 18

 22

 21

 21

 21

 body weight (g)

 100%

 100%

 NS

 

 NS

 

 89.91**

 91.34**

 brain                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 102.5*

 NS

 NS

relative weight (g/100g bw) 

 100%

 100%

 NS

 NS

 NS

 

 110.20**

 110.05**

 thymus                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 81.72**

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 Livera                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 84.80**

 86.24**

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 94.26*

 94.56**

 Kidneya                        

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

 spleen                         

 absolute weight (g)

 100%

 100%

 NS

 NS

 NS

 NS

 86.65**

 84.11

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

ovarya                      

absolute weight (g) 

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 84.52**

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

                       uterusa 

 absolute weight (g)

 100%

 100%

NS

NS 

 83.85*

 NS

 78.47**

 81.49*

 relative weight (g/100g bw)

 100%

 100%

 NS

 NS

 NS

 NS

 NS

 NS

NS- not statistically significant compared to untreated control

**- significantly different from control, p<0.05

*- significantly different from control, p< 0.01

Applicant's summary and conclusion

Conclusions:
Aluminium sulfate had no effect on reproductive performance