Rapal 01, Paval, Serox, Argalum

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

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 guideline

Test guideline 1

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

Test guideline 2

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:

not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium sulphate

EC Number:

233-135-0

EC Name:

Aluminium sulphate

Cas Number:

10043-01-3

Molecular formula:

Al.3/2H2O4S; General formula Al2(OH)x(SO4)(3-x/2), with x=0 and x=3 and x ranging from 0 to 3.

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: AS 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.
- Age at study initiation: (P) 5 wks; (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing:
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dosing solutions 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:

- M/F ratio per cage: 1:1 from the same treatment group
- Length of cohabitation: until successful copulation occured or the mating period of 2 weeks had elapsed
- Proof of pregnancy: during mating period vaginal smears were examined daily for the presence of sperm, presence of sperm in vaginal smears/or vaginal plug were considered as evidence of successful mating (day0 of gestation)
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: after day17 of gestation to day21 after delivery, wire-mesh floors of cages was replaced with stainless steel tray and individual dams or litters were reared using wood chipsas bedding

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

during the study the, the concentrations of AS in drinking water were analyzed in the first and last preparations and once every 3 months, and verified using high performance liquid chromatography (quantitation limit 5 µg/mL)

Duration of treatment / exposure:

F0 males: 10 weeks prior to mating, during mating and up to parturition of the paired females
F0 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 day0 of dosing) and were treated in same way as F0 males and females

Frequency of treatment:

animals were dosed up to the day prior to necropsy

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 (F0)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 ppm (nominal)

Remarks:

For actual doses received see table1 under any other information on materials and methods including tables.

Doses / concentrations 2

Dose / conc.:

600 ppm (nominal)

Remarks:

For actual doses received see table1 under any other information on materials and methods including tables.

Doses / concentrations 3

Dose / conc.:

3 000 ppm (nominal)

Remarks:

For actual doses received see table1 under any other information on materials and methods including tables.

No. of animals per sex per dose:

24 animals per sex and group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on the results of a preliminary range-finding study
- Rationale for animal assignment: by stratified random sampling based on body weight

Positive control:

not required

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 F0 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 epididymalfluid 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 toal count by the gram weight of the caudal epididymis.
Sperm orphology was studied by stainign 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 liter 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 a parturition of their paired females).
- Maternal animals: All surviving animals, after the last litter of each generation was 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 evdence 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 doese 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 sacrifized 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 substnace-related organ weigth 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 atsexual maturation, behavioral 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 fr 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 of 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 F0 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 F0 female died (non adverse); 3000 ppm: one F1 femlae 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 male or female P0 rats.
600 ppm: One P0 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.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Drinking water consumption: P0 males and females - significantly lower than control throughout the study (120-3000 ppm)
Food consumption: P0 males - 600 ppm: significantly reduced during the first week of dosing; 3000 ppm: significantly decreased during weeks 1,8 and 13-14; P0 females - 600 ppm: significantly decreased during week 3 of lactation; 3000 ppm: significantly decreased at 1 week of dosing and during week 3 of lactation;
Body weight: P0 males and females - 3000 ppm: significantly decreased in the first 2 or 3 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)
P0 females - 120, 600 and 3000 ppm:
estrous cycle: no significant deviations in the estrous cycle of F0 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)
P0 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. 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 P0 adults (Note: no details were provided on the results of these examinations).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0 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 table 2)
- no significant differences were observed between the control and AS-treated groups in F0 generation.
Copulation was not observed:
- in the P0 males: control (n=2), 120 ppm (n=2), 3000 ppm (n=2) and in the P0 females: in the control (n=1);
After successful copulation, no pregnancy was observed:
- in P0 females: in the control (n=1), 120 ppm (n=2), 3000 ppm (n=1);
No live pups delivered were found for pregnant rats from :
- P0 female in the 120 (n=1), 600 (n=1) and 3000 ppm (n=1);
Comments: Overall, there were no treatment-related effects on reproduction parameters.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P0 males - 3000 ppm
absolute and relative liver weights
- were significantly decreased;
absolute spleen weight
- was significantly decreased;
- no significant change in relative weight.
P0 females - 120, 600 and 3000 ppm
- no changes in absolute or relative weights of organs compared to the control (data not shown).
P0 females - 3000 ppm
number of primordial follicles in the ovary
- no difference between AS-treated and controls (data not shown).

GROSS PATHOLOGY (PARENTAL ANIMALS)
P0 generation - No dose-related gross lesions were found in P0 adults.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P0 males and females - 3000 ppm:
Histopathological examination of the reproductive organs revealed no compound-related alterations.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

1

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

600 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 8.06 and 13.5 mg Al/kg bw/day in P0 males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: P1 (second parental generation)

Details on results (P1)

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 male or female P1 rats.
120 ppm: One P1 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.
3000 ppm: One P1 male died at 12 weeks of dosing. No clinical signs of toxicity were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Drinking water consumption: P1 males - 120 ppm: significantly decreased during 3-6, 8, 10 weeks of dosing; 600, 3000 ppm: significantly decreased through dosing period; P1 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: P1 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 (P1 females)
Body weight: 120 (P1 males), 600 (P1 males and P1 females) and 3000 ppm (F1 males and F1 females) - no significant differences in body weight compared to control; 120 ppm, P1 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)
P1 females - 120, 600 and 3000 ppm:
estrous cycle: no significant deviations in the estrous cycle of P1 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)
P1 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 F1 adults (Note: no details were provided on the results of these examinations).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
P1 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 table 2)
- no significant differences were observed between the control and AS-treated groups in P1 generation.
Copulation was not observed:
- in the P1 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 P1 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 :
- P1 female in the 120 ppm group (n=1).
Comments: Overall, there were no treatment-related effects on reproduction parameters.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P1 males - 3000 ppm
absolute weight of the adrenals
- was significantly decreased;
- no significant change in relative weight.
P1 males - 600 ppm
absolute weight of the testes
- was significantly decreased;
- no significant change in relative weight.
P1 females - 120, 600 and 3000 ppm
- no changes in absolute or relative weights of organs compared to the control (data not shown).
P1 females - 3000 ppm
number of primordial follicles in the ovary
- no difference between AS-treated and controls (data not shown).

GROSS PATHOLOGY (PARENTAL ANIMALS)
P1 generation - No dose-related gross lesions were found in P1 adults.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P1 males and females - 3000 ppm:
Histopathological examination of the reproductive organs revealed no compound-related alterations.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 38.5 and 55.6 mg Al/kg bw/day in P1 males and females, repsectively

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose tested

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 38.5 and 55.6 mg/kg bw/day in P1 males and females, respectively

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2 F1 showed malformations (non adverse), no malformed F2 pups were observed

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; males - no significant differences

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 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 120 ppm group, but there was no significant difference between the control and AS-treated groups.

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.

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.

GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations:
F1 males and females
- no treatment-related alterations in F1weanlings or in pups found dead during the preweaning period (data not shown).

HISTOPATHOLOGY (OFFSPRING)
F1 males and females
- no dose-related histopathological changes in the liver or spleen of male and female F1 weanlings. (for 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 - 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

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.

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)

1

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

600 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 8.06 and 13.5 mg Al/kg bw/day in P0 males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

3

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively

Sex:

female

Basis for effect level:

sexual maturation

4

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

600 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 8.06 and 13.5 mg Al/kg bw/day in P0 males and females, respectively

Sex:

female

Basis for effect level:

sexual maturation

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

Details on results (F2)

VIABILITY (OFFSPRING)
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)
F2 generation - No malformed F2 pups were found in any group.

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

OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT:
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.
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.
600 ppm - 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
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%).

Effect levels (F2)

1

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Generation:

F2

Effect level:

3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 38.5 and 55.6 mg Al/kg bw/day in P1 males and females, repsectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F2

Effect level:

600 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 9.78 and 14.0 mg Al/kg bw/day in P1 males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F2

Effect level:

>= 3 000 ppm

Based on:

test mat.

Remarks:

Al2(SO4)3; equivalent to 38.5 and 55.6 mg Al/kg bw/day in P1 males and females, repsectively

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose tested

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table2: Reproductive performance of F0 and F1 parental animals

AS (ppm)		0	120	600	3000
F0 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

Table3: 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**
Liver^a
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
Kidney^a
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**
testis^a
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
Epididymis^a
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

Table4: 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
Liver^a
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**
Kidney^a
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
ovary^a
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
uterus^a
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:

Interpretation of the results is difficult due to the clear effect of AS treatment on fluid consumption. Addition of the test substance to drinking water at high concentrations led to reduced pH (3.57 to 4.2) and this appears to have reduced the palatability of the drinking water. At these AS levels, the F0 and F1 females also decreased their food consumption relative to the controls during week 3 of lactation. As a result, due to decreased drinking water consumption and decreased food consumption of F0 and F1 dams during the later stages of lactation, it is not possible to conclude with certainty whether the observations reported were associated with Al or represent secondary effects due to maternal dehydration and reduced nursing that may have influenced pup weight on PND 21. Because the effects reported could be related to decreased maternal fluid consumption, the utility of this study for risk assessment is limited.