Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Published report of a OECD guideline and GLP compliant study, reported in sufficient detail but no full study report available
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The studies cited in IUCLID chapter 7.1.1 (ToxTest 2010; Priest 2010) demonstrate very similar systemic bioavailabilities of a number of aluminium compounds, including aluminium chloride and sulfate. In fact, ECHA has agreed that "a joint assessment of AC, ACH and AS is justified based on read-across" (SEV-D-2114385103 -55 -01/F).
Therefore, read-across from aluminium chloride hexahydrate, basic aluminium chloride and aluminium sulfate was considered appropriate to cover the endpoint of toxicity to reproduction for anhydrous aluminium chloride in accordance with section 1.5 in REACH Annex XI.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Aluminium sulfate, target: aluminium chloride anhydrous

3. ANALOGUE APPROACH JUSTIFICATION
The studies cited in IUCLID chapter 7.1.1 (ToxTest 2010; Priest 2010) demonstrate very similar systemic bioavailabilities of a number of aluminium compounds, including aluminium chloride and sulfate. In fact, ECHA has agreed that "a joint assessment of AC, ACH and AS is justified based on read-across" (SEV-D-2114385103 -55 -01/F).
Therefore, read-across from aluminium chloride hexahydrate, basic aluminium chloride and aluminium sulfate was considered appropriate to cover the endpoint of toxicity to reproduction for anhydrous aluminium chloride in accordance with section 1.5 in REACH Annex XI.
Cross-reference
Reason / purpose for cross-reference:
read-across source
Reference
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
Remarks:
Published report of a OECD guideline and GLP compliant study, reported in sufficient detail but no full study report available
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Specific details on test material used for the study:
AS (CAS No. 10043-01-3) was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). The AS (Lot No. 007X1828) used in this study was 98.5% pure, and was kept
in a sealed container under cool and dark conditions.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Crl:CD(SD) rats (4 weeks old) were purchased from Atsugi Breeding Center, Charles River Laboratories Japan, Inc. (Yokohama, Japan). This strain was chosen
because they are the most commonly used in reproductive and developmental toxicity studies, and historical control data are available. The animals were acclimated
to the laboratory for 7 days, and subjected to treatment at 5 weeks of age. They were carefully observed during the acclimation period, and male and female rats found to be in good health were selected for use. The rats were distributed into four groups of 24 males and 24 females each by stratified random sampling based on body weight, and all animals were assigned a unique number and the ear was tattooed prior to the start of the experiment.
Throughout the study, animals were maintained in an air-conditioned room at 21–25 ◦C, with a relative humidity of 36–59%, a 12-h light/dark cycle (8:00–20:00) and ventilation at 10–15 times/h. They were housed individually, except for the acclimation, mating and nursing periods, in suspended wire-mesh cages. From day 17 of gestation to day 21 after delivery, the wire-mesh floor of the cage was replaced with a stainless-steel tray, and individual dams and litters were reared using wood
chips as bedding (White Flake; Charles River Laboratories Japan, Inc., Yokohama, Japan). All animals were fed ad libitum with a standard rat diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan), but were supplied with different drinking water solutions, as mentioned above, through two generations. Aluminium concentration in the standard diet, analyzed by atomic absorption spectrometry for each lot of diet, ranged from 25ppm to 29 ppm.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
AS (CAS No. 10043-01-3) was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). The AS (Lot No. 007X1828) used in this study was 98.5% pure, and was kept
in a sealed container under cool and dark conditions. The test article was dissolved in ion-exchanged water, and served as drinking water to the animals. Control rats were given the ion-exchanged water alone as drinking water. Before the start of the study, the stability of AS in ion-exchanged water at concentrations of 0.1, 0.6 and 15mg/mL was confirmed after at least 4-day storage at room temperature following 6-day refrigerated storage; therefore, dosing solutions were prepared at least once every 6 days and kept in a cool place until serving. Fresh drinking water was served at least once every 4 days.
Details on mating procedure:
Each female was mated with a single male of the same dosage group until successful copulation occurred or the mating period of 2 weeks had elapsed. For F1
matings, cohabitation of siblings was avoided. During the mating period, vaginal smears were examined daily for the presence of sperm, and the presence of sperm
in the vaginal smear and/or a vaginal plug were considered as evidence of successful mating. The day of successful mating was designated as day 0 of gestation. Females that did not mate successfully during the 2-week mating period were cohabited with another male from the same group who had been proven to copulate with limits of not less than 7 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the study, the concentrations of AS in drinking water were analyzed in the first and last preparations and once every 3 months, and confirmed to be 97.5–106.3% of the target by high performance liquid chromatography. AS contained in the drinking water for the control group was less than the quantitation limit (5µg/mL).
Dose / conc.:
120 ppm (nominal)
Dose / conc.:
600 ppm (nominal)
Dose / conc.:
3 000 ppm (nominal)
Dose / conc.:
8.6 mg/kg bw/day (actual dose received)
Remarks:
F0 males receiving 120 ppm in drinking water
Dose / conc.:
41 mg/kg bw/day (actual dose received)
Remarks:
F0 males receiviving 600 ppm in drinking water
Dose / conc.:
188 mg/kg bw/day (actual dose received)
Remarks:
F0 males receiving 3000 ppm in drinking water
Dose / conc.:
14.4 mg/kg bw/day (actual dose received)
Remarks:
F0 females receiving 120 ppm in drinking water
Dose / conc.:
74.5 mg/kg bw/day (actual dose received)
Remarks:
F0 females receiving 600 ppm in drinking water
Dose / conc.:
316 mg/kg bw/day (actual dose received)
Remarks:
F0 females receiving 3000 ppm in drinking water
Dose / conc.:
10.7 mg/kg bw/day (actual dose received)
Remarks:
F1 males receiving 120 ppm in drinking water
Dose / conc.:
50.2 mg/kg bw/day (actual dose received)
Remarks:
F1 males receiving 600 ppm in drinking water
Dose / conc.:
232 mg/kg bw/day (actual dose received)
Remarks:
F1 males receiving 3000 ppm in drinking water
Dose / conc.:
15.3 mg/kg bw/day (actual dose received)
Remarks:
F1 females receiving 120 ppm in drinking water
Dose / conc.:
74.2 mg/kg bw/day (actual dose received)
Remarks:
F1 females receiving 600 ppm in drinking water
Dose / conc.:
338 mg/kg bw/day (actual dose received)
Remarks:
F1 females receiving 3000 ppm in drinking water
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
Twenty-four F0 rats (5-week-old males and females)/sex/group were exposed to AS in drinking water at 0, 120, 600 or 3000 ppm. After 10-week administration of AS,
each female rat was mated with a male rat of the same dosage group, and pregnant females were allowed to deliver spontaneously and nurse their pups. Administration
of AS was continued throughout the mating, gestation and lactation periods.
F0 parental male rats were necropsied after the parturition of paired females. F0 females were necropsied after weaning of their pups.
For the second (F1) generation, 24 male and 24 female weanlings in each group were selected as F1 parents on PNDs 21–25 to equalize the mean body weights among groups as much as possible. One male and 1 female F1 weanlings were selected from each of litters born during the 5 days including the day of the largest number of F0 parturition, and if the number of litters was insufficient, a second weanling pup in the litter was selected with care to prevent litter effects. 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 were mated, allowed to deliver and nurse their F2 pups, and necropsied in the same manner as described for F0 rats. Unselected F1 weanlings and all F2 weanlings were necropsied on PND 26.
Parental animals: Observations and examinations:
Throughout the study, all parental animals were observed for clinical signs of toxicity at least twice a day. The body weight and food consumption were measured
weekly. For females exhibiting evidence of successful mating, body weight and food consumption were recorded on gestational days 0, 7, 14 and 20 of gestation and
days 0, 7, 14 and 21 of lactation (and additionally day 4 of lactation for body weight). Water consumption was recorded twice a week, and on days 0, 4, 7, 11, 14, 17 and 20 of gestation and days 0, 4, 7, 11, 14, 17, 19 and 21 of lactation. The intake of test substance was calculated based upon mean values for body weight and water
consumption in each group.
Oestrous cyclicity (parental animals):
For each female, daily vaginal lavage samples were evaluated for estrous cyclicity throughout the last 2 weeks of the premating period and during cohabitation
until evidence of copulation was detected. Females having repeated 4–6 day estrous cycles were judged to have normal estrous cycles.
Sperm parameters (parental animals):
Sperm parameters were determined for all F0 and F1 male adults on the day of the scheduled sacrifice. The right testis was used to count testicular
homogenization-resistant spermatid heads. The right epididymal cauda was weighed and used for sperm analysis. For sperm motility, the percentage of motile
sperm and progressively motile sperm, and the swimming speed and pattern were determined using a computer-assisted cell motion analyzer (TOX IVOS; Hamilton
Thorne Bioscience, Beverly, MA, USA). After recording sperm motion, the cauda epididymal fluid was diluted and the sperm were enumerated with a hemacytometer
under a light microscope. Sperm count per gram of epididymal tissue was obtained by dividing the total count by the gram weight of the cauda epididymis. The sperm
was stained with eosin and mounted on a slide glass. Two hundred sperm in each sample were examined under a light microscope, and the percentage of morphologically abnormal sperm was calculated.
Litter observations:
Once insemination was confirmed, female rats were checked at least three times daily on days 21–25 of gestation to determine the time of delivery. The females were allowed to deliver spontaneously and nurse their pups until PND 21 (the day of weaning). The day on which dams held their pups under the abdomen in the nest by 13:00 was designated as day 0 of lactation or PND 0. On PND 0, all live and dead pups were counted, and live pups were sexed and examined grossly. They were observed daily for clinical signs of toxicity, and the body weight of live pups was recorded on PNDs 0, 4, 7, 14 and 21. On PND 4, litters were randomly adjusted to eight pups of four males and four females. No adjustment wasmade for litters of fewer than eight pups. Pups were assigned a unique number and limb tattooed on PND 4.
All F1 and F2 live pups were observed for pinna unfolding from PND 1 to PND 4. Body weight was recorded daily during this period. The anogenital distance (AGD)
was measured using calipers on PND 4 in all F1 and F2 pups, and the normalized value ofAGDto body weight, AGD/cube root of the body weight ratio, was calculated.
One male and one female F1 and F2 pup selected from each dam were evaluated for incisor eruption beginning on PND 8 and eye opening beginning on PND 12, and
continued until each pup fulfilled the criteria. The body weight of the respective F1 and F2 pups was recorded on the day the criteria were fulfilled. Surface righting
reflex, negative geotaxis and mid-air righting reflex were assessed on PND 5, 8 and 18, respectively, for one male and one female F1 and F2 pup selected from each
dam. All F1 offspring selected as F1 parents were observed daily for male preputial separation beginning on PND 35 or female vaginal opening beginning on PND 25
until completion. The body weight of the respective F1 rats was recorded on the day of completion of these pubertal landmarks.
Spontaneous locomotor activity was measured at 4weeks of age in 10 male and 10 female F1 rats randomly selected from each group, using a multi-channel activity
monitoring system (SUPERMEX; Muromachi Kikai Co., Ltd., Tokyo, Japan). Rats were placed individually in transparent polycarbonate cages [285 (W) mm×450
(D) mm×210 (H) mm, CL-0108-1; CLEA Japan, Inc., Tokyo, Japan], which were placed under an infrared sensor that detects thermal radiation from animals, and
spontaneous motor activity was determined at 10-min intervals and for 60 min. A test in a water-filled multiple T-maze was conducted in 10 male and 10 female
F1 rats selected from each group at 6 weeks of age. The apparatus was similar to that described by Biel [45]. The water temperature of the maze was kept 20.5–22 ◦C. As a preliminary swimming ability test, each rat was allowed to swim three times in a straight channel on the day before the maze trial, and then tested in the maze with
three trials per day for the next three consecutive days. The elapsed time between entry into the water at the starting point and touching the goal ramp, and thenumber
of errors were recorded. To prevent the exhaustion of the rats, no animalwasallowed to remain in the water for more than 3min in any trial.
Postmortem examinations (parental animals):
All surviving parental male rats were euthanized by exsanguination under ether anesthesia after the parturition of paired females. All female rats showing successful
reproductive performance were evaluated for estrous cycle stage by examination of the vaginal smear after weaning of pups, and euthanized at the proestrous stage
by exsanguination under ether anesthesia. Females that did not copulate or had not completed parturition and dams with total litter loss were euthanized in the same way around the same time as females with successful reproduction. For all parental animals, the external surfaces were examined. The abdomen and thoracic cavity were opened, and gross internal examination was performed. Major organs were removed and the number of uterine implantation sites was recorded for each female. The testis and epididymis were fixed with Bouin’s solution and preserved in 70% ethanol, and the other organs were stored in 10% neutral-buffered formalin.
The brain, pituitary, thyroids, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, seminal vesicles (with coagulating glands and their fluids), ventral
prostate, uterus and ovaries were weighed before fixation. The thyroid and seminal vesicle were weighed after fixation.
Histopathological evaluations were performed in all animals of the control and highest dose groups, in females with abnormal estrous cycles, abnormal delivery or totally dead pups, in males and females without evidence of copulation or insemination, and in all animals with grossly abnormal reproductive organs. Of these animals, the testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina, which were fixed as mentioned above, were embedded in paraffin by a routine procedure. They were sectioned, stained with hematoxylin–eosin and examined histopathologically under a light microscope. If treatment-related histopathological changes were found in the highest dose group, were the same tissues from the next lower dose group then examined. In 10 F1 females, randomly selected from the control and highest dose groups, the number of primordial follicles was counted as follows. The right ovary, fixed in 10% neutral-buffered formalin, was dehydrated and then embedded in paraffin in longitudinal orientation by routine procedures. Sections were cut serially at 5m and every 20th section was serially mounted on a slide and stained with hematoxylin and eosin. About 40 sections per ovary were used to determine the primordial follicles.
Postmortem examinations (offspring):
Following the adjustment of litter size on PND4, culled pups were euthanized by inhalation of carbon dioxide and subjected to a gross external and internal
observation. Grossly abnormal organs/tissues were removed and stored in 10% neutral-buffered formalin. All pups found dead before weaning were necropsied
immediately, and the whole body was stored in 10% neutral-buffered formalin.
F1 weanlings not selected to become parents and all F2 weanlings were euthanized and necropsied on PND 26, as described for adults. For one male and one
female F1 and F2 weanlings selected from each dam, the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries
were removed and the organ weights were measured. Major organs, including the weighed organs, were stored in 10% neutral-buffered formalin.
Since test substance-related organ weight changes were found in the liver and spleen of the highest dose group, they were histopathologically examined for 10
male and 10 female F1 and F2 weanlings in the control and highest dose groups. The examined animals were randomly selected from animals whose organs were stored. If treatment-related histopathological changes were observed in the highest dose group, were the same tissues from the next lower dose group then examined. For the histopathological examination, paraffin sections were routinely prepared and stained with hematoxylin and eosin.
Statistics:
Parametric data, such as body weight, food and water consumption, length of the estrous cycle and gestation, precoital interval, the number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, parameters of behavioral tests, organ weight and sperm parameters, were analyzed by Bartlett’s test for homogeneity of distribution. For preweaning pups, body weight, AGD, viability, and age at the completion of developmental landmarks were similarly analyzed using the litter as the experimental unit. When homogeneity was recognized, oneway analysis of variance was performed. If a significant difference was detected, Dunnett’s test was conducted for comparisons between control and individual treatment groups. Data without homogeneity were analyzed using the Kruskal–Wallis rank sum test. If significant differences were found, the Mann Whitney’s U test was conducted for comparison between the control and each dosage group. The incidence of parental animals with clinical signs, and autopsy and histopathological findings, the incidence of females with normal estrous cycles, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ratio and completion rate of negative geotaxis were compared between the AS and control group using Fisher’s exact test. The incidence of pups with clinical signs or autopsy findings per litter, the completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex were analyzed by the Wilcoxon rank sum test. The number of primordial follicles in the control and highest dose groups was compared by Student’s t-test because the homogeneity of variance was indicated by the F-test. All of these statistical analyses were conducted using the 5% level of probability as the criterion for significance.
Clinical signs:
no effects observed
Description (incidence and severity):
No significant difference was seen between control and AS-treated groups in the incidence of clinical signs of toxicity in either male or female F0 and F1 rats
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the 600ppm group, a subcutaneous mass was observed in the abdominal region of one F0 female from the beginning of 5 weeks of dosing, and this animal was found dead at 2 weeks of gestation. No abnormality was found on gross internal examination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of F0 males and females was significantly lowered in the first 2 or 3 weeks of dosing at 3000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In F0 males, there were significant decrease in food consumption in the first week of dosing at 600 and 3000 ppm, and during week 8 and weeks 13–14 of dosing at 3000 ppm. Food consumption of F0 females showed a significantly lower value during week 1 of dosing at 3000ppm and during week 3 of lactation at 600 and 3000 ppm
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In F0 males and females of all AS-treated groups, water consumption was significantly lower than in controls almost throughout the dosing period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination of the reproductive organs revealed no compound-related alterations.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
During the premating period, AS produced no significant deviations in the estrous cycle of F0 and F1 females although a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
As for the sperm parameters examined for scheduled sacrificed adults, in F0 generation, the absolute number of cauda epididymal sperm was significantly decreased at 3000ppm (253.8±61.3×106/cauda versus 286.3±40.3×106/cauda in the control); however, no significant changes were found in the number per gram of tissue. No such change was observed in F1 adults. There were no significant differences in the number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm between control and AS-treated groups in either F0 or F1 adults.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
During the mating period, copulation was not observed in two males each in the control, 120ppm and 3000ppm groups and in one female of the control group in the F0 generation.
Among females with successful copulation, one female each in the control and 3000ppm group and two females at 120ppm in the F0 generation were not impregnated.
In addition, one pregnant F0 female each at 120, 600 and 3000ppm did not deliver live pups; however, there were no significant differences in the copulation, fertility or gestation index, and the precoital interval or gestation length between the control and AS-treated groups in F0 and F1 generation.
No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.
Dose descriptor:
LOAEL
Effect level:
188 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
316 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Clinical signs:
no effects observed
Description (incidence and severity):
No significant difference was seen between control and AS-treated groups in the incidence of clinical signs of toxicity in either male or female F0 and F1 rats
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In the 120ppm group, one F1 male was found dead at 9 weeks of dosing. In this animal, soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. autopsy, various changes, including accumulation of ascitic and pleural fluid and dark purple discoloration of the liver and kidneys, were found.
One F1 male at 3000ppm was also found dead at 12 weeks of dosing without any clinical signs of toxicity. No abnormality was found on gross internal examination.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight of F1 males and females exhibited no significant differences between the control and AS-treated groups, except that F1 females of the 120ppm group had significantly higher body weight during weeks 6–8 of dosing.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly decreased during week 10 of dosing in F1 males of the 600 and 3000ppm groups, and during week 3 of lactation in F1 females of the same groups. There was also a transient significant increase in food consumption during week 6 of dosing in F1 females of the 120ppm group.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was significantly decreased through the dosing period in 600ppm and 3000ppm treated males, and during weeks 3–6, week 8 and week 10 of dosing in 120ppm treated males. In F1 females, significant reductions in water consumption were found almost throughout the dosing period at 3000 ppm, during week 10 of dosing and week 3 of lactation at 600 ppm, and during weeks 9–10 of dosing at 120 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In F1 males, the absolute weights of the adrenals at 3000ppm and the testes at 600ppm were significantly decreased without significant changes in the relative weight. There were no significant changes in the absolute and relative weights of any organ in F0 and F1 female adults
Gross pathological findings:
no effects observed
Description (incidence and severity):
No dose-related gross lesions were found in F0 or F1 adults.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination of the reproductive organs revealed no compound-related alterations. There was no significant difference
in the number of primordial follicles in the ovary of F1 females between control and 3000ppm groups.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
During the premating period, AS produced no significant deviations in the estrous cycle of F0 and F1 females although a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no significant differences in the number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm between control and AS-treated groups in either F0 or F1 adults.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
In the F1 generation, one male in the control group, two males and one female in the 120ppm group, one male in the 600ppm group, and three males and one female in the 3000ppm group did not copulate.
Among females with successful copulation, two females each in the control, 600ppm and 3000ppm groups, and four females at 120ppm in the F1 generation were not impregnated. In addition, one pregnant F1 female at 120ppm did not deliver live pups; however, there were no significant differences in the copulation, fertility or gestation index, and the precoital interval or gestation length between the control and AS-treated groups in F0 and F1 generation.
No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.
Dose descriptor:
LOAEL
Effect level:
232 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
338 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant changes were found in the sex ratio of pups and the viability index in either generation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 3000ppm group, the body weight of male and female F1 pups was significantly lower than the control on PND 21.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
As for the sexual development of F1 male and female animals, vaginal opening was significantly delayed at 3000ppm (31.4±1.7, compared to 29.5±2.1 in control). At this dose, body weight at the time of vaginal opening was slightly heavier than the control (119.0±13.3 g versus 109.6±11.6 g) although not statistically significant. No significant differences between control and AS-treated groups were noted in the age at preputial separation or body weight at the time of completion in males.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the 3000 ppm group, absolute and relative liver weights were significantly lower than the controls. Absolute spleen weight was also decreased significantly in both sexes of the 3000ppm group, accompanied by a significant decrease in the relative weight in males. In addition, significant decreases in the absolute weight were found for the thymus in both sexes and for the kidneys, testes and epididymides in males at 3000 ppm, and for the uterus in females at 600 and 3000 ppm. Relative brain weight was significantly increased in both sexes of the 3000ppm group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal gross observations revealed no compound-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Spontaneous locomotor activity at 10-min intervals and for 60 min was not significantly different between control and AStreated groups in male and female F1 rats. In the water-filled T-maze test, pre-test swimming trials in the straight channel revealed that all male and female F1 rats in each group could swim satisfactorily, and no significant changes were observed in the elapsed time to traverse the straight channel. On days 2–4 of the T-maze test, no significant changes were observed in the elapsed time and number of errors in males. In females, the elapsed time and the number of errors on day 2 of the T-maze was significantly lowered at 600 ppm, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 of the T-maze test between control and AS-treated groups.
Gross examination of delivered pups revealed one F1 pup with trauma in the perianal region and tail in the control group and one F1 pup with hemimelia and oligodactyly in the 120ppm group, but no significant difference was found in the incidence between the control and AS-treated groups.
For the physical development of male and female F1 pups and male F2 pups, there was no significant difference in the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening between the control and AS-treated groups.
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.
All male and female F1 pups in all groups achieved the surface righting reflex onPND5, negative geotaxis reflex onPND8 and midair righting reflex on PND 18. No significant changes were observed in the response time of surface righting and negative geotaxis reflex.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of F2 female pups were significantly lower than controls on PND 21 at 3000 ppm. There were no significant differences in the body weight of male F2 pups between the control and AS-treated groups during the preweaning period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight at scheduled sacrifice was significantly lowered in both sexes of the 3000ppm group. In males, the absolute and relative weights of the thymus and spleen were significantly decreased in the 3000ppm group. Significant decreases were also found in the absolute weight of the liver and epididymides at 3000 ppm. The relative brain weight was significantly increased at this dose. At 120 ppm, the only significant changewasa non-dose-related decrease in the relative thymus weight. In F2 females, there were significant decreases in the absolute and relative weights of the liver, and the absolute weight of the spleen, ovary and uterus, and a significant increase in the relative brain weight at 3000 ppm. In addition, a significant decrease in the absolute brain weight was observed only in the 600ppm group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal gross observations revealed no compound-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no dose-related histopathological changes in the liver and spleen of male and female F1 and F2 weanlings.
For the physical development of male and female F1 pups and male F2 pups, there was no significant difference in the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening between the control and AS-treated groups.
In female F2 pups, the completion rate of pinna unfolding on PND 2 was significantly lower in the 600ppm group (17.0±35.4%, compared with 45.8±46.9 in controls), but no dose dependency was observed in this change. No significant changes were found in the completion rate of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks in female F2 pups.
The AGD and AGD per cube root of the body weight ratio were not significantly different between control and AS-treated groups in male and female F1 and F2 pups.
In F2 pups, one female of the 600ppm group failed in one of three trials of the mid-air righting reflex on PND 18; however, there was no significant difference in the mean success rate between the control and 600ppm groups (100±0.0% versus 98.4±7.3%). The surface righting reflex on PND 5 and negative geotaxis reflex on PND 8 were achieved in all male and female F2 pups in all groups, and no significant changes were found in the response time.
Dose descriptor:
LOAEL
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Reproductive effects observed:
no

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

from drinking water and food combined was estimated from the water and food consumption and body weight. Average

aluminium intake was 1.62, 2.96, 8.06 and 31.2mg Al/kg bw/day in F0 males, 2.29, 4.50, 13.5 and 52.0mg Al/kg bw/day in F0 females, 1.93, 3.55, 9.78 and 38.5mg Al/kg bw/day in F1 males, and 2.35, 4.72, 14.0 and 55.6mg Al/kg bw/day in F1 females for control through high-dose groups.

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 (%)a  males  91.7  91.7  100  91.7
   females  95.8  100  100  100
 Precoital interval (days)b    3.2 ± 1.1  3.2 ± 1.8  2.9 ± 1.3  2.8 ± 1.6
 Fertility index (%)c  males  95.5  90.9  100  95.5
   females  95.7  91.7  100  95.8
 Gestation index (%)d    100  95.5  95.7  95.7
 Gestation length (days)b  22.4 ± 0.5  22.5 ± 0.6  22.1 ± 0.4  22.3 ± 0.5
 Delivery index (%)b,e    94.3 ± 5.6  88.6 ± 21.0  90.7 ± 20.8  92.0 ± 20.5
 F1 generation          
 No. of rats (male/female)    24/24  23/24  24/24  24/24
 Copulation index (%)a  males  95.8  91.3  95.8  87.5
   females  100  95.8  100  95.8
 Precoital interval (days)b    3.3 ± 3.2  3.0 ± 2.0  2.7 ± 1.5  2.3 ± 1.1
 Fertility index (%)c  males  91.3  81.0  91.3  95.2
   females  91.7  82.6  91.7  91.3
 Gestation index (%)d    100  94.7  100  100
 Gestation length (days)b    22.4 ± 0.5  22.3 ± 0.5  22.2 ± 0.4  22.2 ± 0.4
 Delivery index (%)b,e    94.0 ± 9.9  87.5 ± 22.6  91.4 ± 10.7  94.6 ± 6.8

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

b Values are given as the mean±S.D.

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

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

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

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

AS (ppm)

0 (control)

120

600

3000

F1 offspring

 

 

 

 

No. of litters

22

21

22

22

No. of pups delivereda

13.9±1.7

12.4±4.7

13.1±4.1

13.1±3.4

Sex ratio of pupsb

0.503

0.462

0.513

0.536

Viability index of pups (%)a

 

 

 

 

On PND 0c

100.0±0.0

99.3±2.3

99.7±1.6

99.5±2.4

On PND 4d

98.7±2.9

95.2±21.8

98.8±2.6

98.0±5.4

On PND 21e

99.4±2.7

100.0±0.0

100.0±0.0

99.4±2.7

Male pup weight during lactation (g)a

 

 

 

 

On PND 0

7.05±0.61

7.25±0.99

6.74±0.69

6.96±0.76

On PND 4

11.04±0.85

11.41±1.99

10.86±1.37

11.00±1.06

On PND 7

18.91±1.29

19.36±2.77

18.59±1.71

18.47±1.35

On PND 14

37.7±2.63

37.97±3.08

37.39±2.59

36.34±2.41

On PND 21

62.48±4.5

62.63±6.14

60.77±4.01

57.34±4.86**

Female Pup weight during lactation (g)a

 

 

 

 

On PND 0

6.61±0.55

6.89±0.83

6.35±0.57

6.60±0.64

On PND 4

10.46±0.89

11.06±1.71

10.27±1.33

10.43±0.83

On PND 7

18.03±1.27

18.56±2.31

17.69±1.61

17.61±1.21

On PND 14

36.29±2.71

36.94±3.03

35.67±2.60

35.31±2.24

On PND 21

60.17±4.16

60.87±5.68

57.68±4.33

55.60±4.34**

F2 offspring

 

 

 

 

No. of litters

22

18

22

21

No. of pups delivereda

13.1±3.6

13.2±3.8

12.6±3.9

14.0±1.9

Sex ratio of pupsb

0.528

0.502

0.536

0.457

Viability index of pups (%)a

 

 

 

 

On PND 0c

99.68±1.51

99.49±2.14

98.42±3.57

98.69±3.60

On PND 4d

94.72±14.54

98.07±5.45

99.07±3.15

99.01±2.49

On PND 21e

100.00±0.00

98.61±4.04

100.00±0.00

100.00±0.00

Male pup weight during lactation (g)a

 

 

 

 

On PND 0

6.97±0.68

6.92±0.81

6.87±0.74

6.89±0.60

On PND 4

10.73±1.62

10.53±1.27

11.27±1.81

10.52±1.15

On PND 7

17.96±2.05

17.51±2.12

18.83±2.39

17.72±1.60

On PND 14

35.79±3.52

36.18±3.63

37.32±4.15

35.44±2.73

On PND 21

59.61±5.45

59.44±5.67

60.12±7.12

56.36±4.47

Female Pup weight during lactation (g)a

 

 

 

 

On PND 0

6.66±0.69

6.38±0.78

6.41±0.65

6.50±0.49

On PND 4

10.22±1.63

9.70±1.23

10.36±1.54

9.98±0.91

On PND 7

17.03±1.99

16.36±2.35

17.40±2.18

16.89±1.23

On PND 14

34.82±3.52

34.17±3.58

34.96±4.24

34.01±2.09

On PND 21

57.33±4.90

56.11±5.54

56.41±6.04

54.16±2.82*

a Values are given as the mean±S.D.

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

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

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

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

* Significantly different from the control, P < 0.05.

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

Absolute and relative organ weight of F1 male and female weanlings.

AS (ppm)

 

0 (control)

120

600

3000

Males

 

 

 

 

 

No. of animals

 

22

20

22

22

Body weight

(g)

90.8±6.9

93.4±10.5

89.7±6.1

79.4±7.5**

Brain

(g)

1.69±0.06

1.73±0.08

1.72±0.07

1.68±0.05

 

(g/100 g bw)

1.88±0.13

1.87±0.19

1.92±0.09

2.14±0.17**

Thymus

(mg)

375±55

384±86

357±58

305±51**

 

(mg/100 g bw)

414±56

409±64

398±59

383±36

Liver

(g)

4.33±0.43

4.40±0.60

4.22±0.45

3.49±0.53**

 

(g/100 g bw)

4.77±0.30

4.71±0.33

4.70±0.27

4.37±0.30**

Kidneya

(g)

1.06±0.09

1.09±0.14

1.03±0.11

0.95±0.13**

 

(g/100 g bw)

1.17±0.06

1.16±0.07

1.15±0.08

1.20±0.07

Spleen

(mg)

394±49

410±68

388±74

301±43**

 

(mg/100 g bw)

436±63

437±40

432±73

379±37**

Testisa

(mg)

596±65

583±67

569±65

539±51*

 

(mg/100 g bw)

657±64

626±49

635±64

682±58

Epididymisa

(mg)

81.8±8.6

76.8±10.9

76.5±8.4

72.0±9.9**

 

(mg/100 g bw)

90.4±10.3

82.0±6.1

85.4±8.4

91.5±14.6

Females

 

 

 

 

 

No. of animals

 

22

20

22

21

Body weight

(g)

84.3±6.3

85.9±9.2

80.5±7.0

75.8±6.4**

Brain

(g)

1.64±0.06

1.66±0.06

1.63±0.05

1.63±0.07

 

(g/100 g bw)

1.96±0.12

1.95±0.18

2.04±0.17

2.16±0.14**

Thymus

(mg)

383±66

373±74

345±46

313±33**

 

(mg/100 g bw)

453±63

433±64

429±57

415±41

Liver

(g)

3.83±0.47

3.92±0.48

3.61±0.35

3.24±0.34**

 

(g/100 g bw)

4.53±0.30

4.57±0.31

4.48±0.30

4.27±0.25*

Kidneya

(g)

0.99±0.11

0.99±0.09

0.93±0.10

0.93±0.10

 

(g/100 g bw)

1.17±0.08

1.15±0.07

1.15±0.09

1.23±0.09

Spleen

(mg)

337±62

356±55

341±64

292±43*

 

(mg/100 g bw)

400±67

415±44

422±53

386±47

Ovarya

(mg)

25.3±4.8

25.3±3.8

22.5±4.6

24.7±3.2

 

(mg/100 g bw)

30.1±5.1

29.7±5.0

27.9±5.0

32.5±4.2

Uterus

(mg)

70.6±16.6

74.2±32.0

59.2±11.9*

55.4±13.4**

 

(mg/100 g bw)

83.8±19.2

85.5±32.4

73.3±11.9

73.3±18.0

Values are given as the mean±S.D.

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

* Significantly different from the control, P < 0.05.

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

Absolute and relative organ weight of F2 male and female weanlings.

AS (ppm)

 

0 (control)

120

600

3000

Males

 

 

 

 

 

No. of animals

 

21

18

22

21

Body weight

(g)

87.7±5.8

89.0±8.7

87.0±9.6

79.2±6.8**

Brain

(g)

1.66±0.05

1.69±0.06

1.70±0.06

1.67±0.06

 

(g/100 g bw)

1.90±0.13

1.91±0.17

1.97±0.16

2.13±0.17**

Thymus

(mg)

382±50

348±49

357±66

305±36**

 

(mg/100 g bw)

439±70

392±52*

411±57

386±40**

Liver

(g)

3.93±0.37

4.04±0.64

3.91±0.39

3.45±0.41**

 

(g/100 g bw)

4.49±0.34

4.52±0.44

4.50±0.24

4.36±0.23

Kidneya

(g)

1.02±0.09

1.01±0.13

0.99±0.13

0.94±0.10

 

(g/100 g bw)

1.16±0.08

1.14±0.06

1.14±0.07

1.19±0.06

Spleen

(mg)

368±54

381±62

361±49

296±48**

 

(mg/100 g bw)

421±64

427±50

416±48

372±42**

Testisa

(mg)

559±67

549±98

543±77

534±54

 

(mg/100 g bw)

637±60

615±81

624±47

680±92

Epididymisa

(mg)

75.3±6.9

78.3±8.8

75.1±10.7

70.5±5.7*

 

(mg/100 g bw)

86.1±8.3

88.4±9.0

86.5±9.0

89.4±8.2

Females

 

 

 

 

 

No. of animals

 

22

18

21

21

Body weight

(g)

80.8±6.0

80.0±7.2

80.8±9.1

73.8±4.4**

Brain

(g)

1.60±0.06

1.61±0.05

1.64±0.05*

1.61±0.04

 

(g/100 g bw)

1.99±0.14

2.03±0.16

2.05±0.20

2.19±0.15**

Thymus

(mg)

337±45

364±36

347±49

312±37

 

(mg/100 g bw)

419±61

457±50

431±47

424±54

Liver

(g)

3.56±0.35

3.61±0.39

3.61±0.48

3.07±0.26**

 

(g/100 g bw)

4.41±0.21

4.51±0.26

4.47±0.26

4.17±0.29**

Kidneya

(g)

0.95±0.07

0.93±0.10

0.92±0.10

0.88±0.08

 

(g/100 g bw)

1.18±0.08

1.16±0.09

1.14±0.06

1.20±0.07

Spleen

(mg)

320.9±46.7

331.8±59.3

331.3±57.1

269.9±55.2*

 

(mg/100 g bw)

398.4±59.0

414.8±64.3

409.0±42.2

365.0±67.4

Ovarya

(mg)

23.9±3.7

22.8±3.6

23.2±3.5

20.2±2.3**

 

(mg/100 g bw)

29.7±4.9

28.8±5.6

29.0±4.7

27.5±3.5

Uterus

(mg)

60.5±17.0

63.8±18.4

65.0±41.7

49.3±11.6*

 

(mg/100 g bw)

74.6±19.2

79.3±19.3

78.7±40.4

67.0±16.2

Values are given as the mean±S.D.

aValues represent the total weights of the organs on both sides.

* Significantly different from the control, P < 0.05.

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

Conclusions:
Continuous drinking of AS-contained water for two generations did not result in changes in copulation, fertility or gestation indices, pre-coital or gestation length, the number of implantations or pups delivered, or the incidence of pups with malformations or variations. In addition, adverse effects were not found in estrous cyclicity or sperm parameters, and the histopathology of reproductive tissues in male and female parental animals.
The most prominent effect of AS administered via drinking water was decreased water consumption. This was likely a consequence of acidification of the AS containing dosing solution to pH 3.57-4.2, which probably reduced the palatability of the drinking water in the AS-treated groups. Decreased water consumption was associated with decreased food consumption by F0 and F1 males and females in the 600 and 3000 ppm group. Since water-deprived animals typically reduce their levels of feed consumption and consequently lower their body weight, decreased food consumption and body weight observed in the present study could be considered secondary to the decreased water consumption.
Male and female F1 pups and female F2 pups in the 3000ppm group had a lower body weight on PND 21 while no difference was found in the birth weight. In F1 and F2 weanlings, organ weight changes were found in the 3000ppm group. Among them, an increase in the relative brain weight is considered to be a secondary change that occurs with the fall in body weight because the absolute weight did not change. Similarly, decreased absolute weights of the kidneys, testes, epididymides, uterus, etc., without changes in the relative weight, were thought to be associated with decreased body weight. On the other hand, the effects on the liver and spleen, the absolute and relative weights of which were decreased in both generations, could not be explained only by the fall of body weight. However, no histopathological changes were detected in the liver and spleen. Furthermore, the changes in the liver or spleen weight were not detected in adults, except for F0 males in the 3000ppm group; therefore, organ weight changes observed in F1 and F2 weanlings in the 3000ppm group were not deemed to be adverse effects. Organ weight changes in the 120 and 600ppm groups were not considered to have toxicological significance because these changes were not dose-dependent or were inconsistent across generations.
As these offspring effects were mainly associated with reduced water and food consumption, they may be attributed to maternal dehydration and corresponding reduced nursing capacity, rather than to aluminium exposure.
As for effects on the developmental landmarks, vaginal opening was slightly delayed in F1 females in the 3000ppm group while no compound-related changes were found in the other developmental landmarks, including male preputial separation. The delay in vaginal opening was not accompanied by adverse changes in estrous cyclicity, AGD or other hormone-dependent parameters. These observations were likely secondary to the decreased body weight development.
In conclusion, no Al-dependent effect on reproduction can be deduced from the study. The authors concluded that their NOAEL of 600 ppm was conservative.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes

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
Test material form:
not specified
Details on test material:
no data

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
188 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
316 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake

Results: P1 (second parental generation)

Effect levels (P1)

open allclose all
Dose descriptor:
LOAEL
Effect level:
232 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
338 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake

Results: F1 generation

Effect levels (F1)

Dose descriptor:
LOAEL
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Results: F2 generation

Effect levels (F2)

Dose descriptor:
LOAEL
Effect level:
3 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion


Route: .live2