Aluminium potassium bis(sulphate)

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented study according to OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2011

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

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

IUPAC Name:

aluminium sulphate

Details on test material:

Aluminium sulfateThe substance was obtained from Kanto Chemical Co., Inc. (Tokyo,Japan). The substance (Lot No. 007X1828) used in this study was 98.5% pure.

Test animals

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Temperature: 21-25 °CRelative Humidity: 36-59%12h-dark/light cycleAir-conditioned room: ventilation at 10–15 times/h

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on mating procedure:

Each female was mated with a single male of the same dosage group until successfulcopulation occurred or the mating period of 2 weeks had elapsed. For F1matings, cohabitation of siblings was avoided. During the mating period, vaginalsmears were examined daily for the presence of sperm, and the presence of spermin the vaginal smear and/or a vaginal plug were considered as evidence of successfulmating. The day of successful mating was designated as day 0 of gestation. Femalesthat did not mate successfully during the 2-week mating period were cohabited withanother male from the same group who had been proven to copulate with limits ofnot less than 7 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The substance 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 the substance in ion-exchanged water at concentrations of 0.1, 0.6 and 15mg/mLwas confirmed after at least 4-day storage at room temperature following 6-dayrefrigerated storage; therefore, dosing solutions were prepared at least once every6 days and kept in a cool place until serving. Fresh drinking water was served at leastonce every 4 days. During the study, the concentrations of the substance in drinking water wereanalyzed in the first and last preparations and once every 3 months, and confirmedto be 97.5–106.3% of the target by high performance liquid chromatography. The substancecontained in the drinking water for the control group was less than the quantifiedlimit (5µg/mL).

Duration of treatment / exposure:

Dose finding study:Males were dosed for 7 weeks, beginning 14 days before mating, and females were dosed for 6–8 weeks beginning14 days before mating to day 4 of lactation throughout the mating and gestation period.Main study:Twenty-four F0 rats (5-week-old males and females)/sex/group were exposed toAluminium sulfate (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 pregnantfemales were allowed to deliver spontaneously and nurse their pups. Administrationof AS was continued throughout the mating, gestation and lactation periods.F0 parental male rats were necropsied after the parturition of paired females. F0females were necropsied after weaning of their pups.For the second (F1) generation, 24 male and 24 female weanlings in each groupwere selected as F1 parents on PNDs 21–25 to equalize the mean body weightsamong groups as much as possible. One male and 1 female F1 weanlings wereselected from each of litters born during the 5 days including the day of the largestnumber of F0 parturition, and if the number of litters was insufficient, a secondweanling pup in the litter was selected with care to prevent litter effects. The day onwhich F1 parental animals were selected was designated as day 0 of dosing for the F1generation. F1-selected rats were given drinking water with the respective formulation,and were mated, allowed to deliver and nurse their F2 pups, and necropsiedin the same manner as described for F0 rats. Unselected F1 weanlings and all F2weanlings were necropsied on PND 26.

Frequency of treatment:

as fresh water every 4 days

Doses / concentrations

Remarks:

Doses / Concentrations:120, 600, 3000 ppmBasis:analytical conc.

No. of animals per sex per dose:

Twenty-four F0 rats (5-week-old males and females)/sex/group

Control animals:

yes

Examinations

Parental animals: Observations and examinations:

Throughout the study, all parental animals were observed for clinical signs oftoxicity at least twice a day. The body weight and food consumption were measuredweekly. For females exhibiting evidence of successful mating, body weight and foodconsumption were recorded on gestational days 0, 7, 14 and 20 of gestation anddays 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 and20 of gestation and days 0, 4, 7, 11, 14, 17, 19 and 21 of lactation. The intake oftest substance was calculated based upon mean values for body weight and waterconsumption in each group.

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Sperm parameters were determined for all F0 and F1 male adults on theday of the scheduled sacrifice. The right testis was used to count testicularhomogenization-resistant spermatid heads. The right epididymal cauda wasweighed and used for sperm analysis. For sperm motility, the percentage of motilesperm and progressively motile sperm, and the swimming speed and pattern weredetermined using a computer-assisted cell motion analyzer (TOX IVOS; HamiltonThorne Bioscience, Beverly, MA, USA). After recording sperm motion, the cauda epididymalfluid was diluted and the sperm were enumerated with a hemacytometerunder a light microscope. Sperm count per gram of epididymal tissue was obtainedby dividing the total count by the gram weight of the cauda epididymis. The spermwas stained with eosin and mounted on a slide glass. Two hundred sperm in eachsample were examined under a light microscope, and the percentage of morphologicallyabnormal sperm was calculated.

Litter observations:

Once insemination was confirmed, female rats were checked at least three timesdaily on days 21–25 of gestation to determine the time of delivery. The females wereallowed to deliver spontaneously and nurse their pups until PND 21 (the day ofweaning). The day on which dams held their pups under the abdomen in the nest by13:00 was designated as day 0 of lactation or PND 0. On PND 0, all live and dead pupswere counted, and live pups were sexed and examined grossly. They were observeddaily for clinical signs of toxicity, and the body weight of live pups was recorded onPNDs 0, 4, 7, 14 and 21. On PND 4, litters were randomly adjusted to eight pups offour males and four females. No adjustment was made for litters of fewer than eightpups. Pups were assigned a unique number and limb tattooed on PND 4.

Postmortem examinations (parental animals):

All surviving parental male rats were euthanized by exsanguination under etheranesthesia after the parturition of paired females. All female rats showing successfulreproductive performance were evaluated for estrous cycle stage by examination ofthe vaginal smear after weaning of pups, and euthanized at the proestrous stageby exsanguination under ether anesthesia. Females that did not copulate or hadnot completed parturition and dams with total litter loss were euthanized in thesame way around the same time as females with successful reproduction. For allparental animals, the external surfaces were examined. The abdomen and thoraciccavity were opened, and gross internal examination was performed. Major organswere removed and the number of uterine implantation sites was recorded for eachfemale. The testis and epididymis were fixed with Bouin’s solution and preservedin 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), ventralprostate, uterus and ovaries were weighed before fixation. The thyroid and seminalvesicle were weighed after fixation.Histopathological evaluations were performed in all animals of the control andhighest dose groups, in females with abnormal estrous cycles, abnormal delivery ortotally dead pups, in males and females without evidence of copulation or insemination,and in all animals with grossly abnormal reproductive organs. Of theseanimals, the testes, epididymides, seminal vesicles, ventral prostate, coagulatinggland, ovaries, uterus and vagina, which were fixed as mentioned above, wereembedded in paraffin by a routine procedure. They were sectioned, stained withhematoxylin–eosin and examined histopathologically under a light microscope. Iftreatment-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, fixedin 10% neutral-buffered formalin, was dehydrated and then embedded in paraffinin longitudinal orientation by routine procedures. Sections were cut serially at5µm and every 20th section was serially mounted on a slide and stained withhematoxylin and eosin. About 40 sections per ovary were used to determine theprimordial follicles.

Postmortem examinations (offspring):

Following the adjustment of litter size on PND4, culled pups were euthanizedby inhalation of carbon dioxide and subjected to a gross external and internalobservation. Grossly abnormal organs/tissues were removed and stored in 10%neutral-buffered formalin. All pups found dead before weaning were necropsiedimmediately, and the whole body was stored in 10% neutral-buffered formalin.F1 weanlings not selected to become parents and all F2 weanlings were euthanizedand necropsied on PND 26, as described for adults. For one male and onefemale F1 and F2 weanlings selected from each dam, the brain, thymus, liver, kidneys,spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovarieswere removed and the organ weights were measured. Major organs, including theweighed organs, were stored in 10% neutral-buffered formalin.Since test substance-related organ weight changes were found in the liver andspleen of the highest dose group, they were histopathologically examined for 10male and 10 female F1 and F2 weanlings in the control and highest dose groups. Theexamined animals were randomly selected from animals whose organs were stored.If treatment-related histopathological changes were observed in the highest dosegroup, were the same tissues from the next lower dose group then examined. Forthe histopathological examination, paraffin sections were routinely prepared andstained with hematoxylin and eosin.

Statistics:

Parametric data, such as body weight, food and water consumption, length of theestrous cycle and gestation, precoital interval, the number of implantations and pupsborn, delivery index, reflex response time, age at sexual maturation, parameters ofbehavioral tests, organ weight and sperm parameters, were analyzed by Bartlett’stest for homogeneity of distribution. For preweaning pups, body weight, AGD, viability,and age at the completion of developmental landmarks were similarly analyzedusing the litter as the experimental unit. When homogeneity was recognized, onewayanalysis of variance was performed. If a significant difference was detected,Dunnett’s test was conducted for comparisons between control and individual treatmentgroups. Data without homogeneity were analyzed using the Kruskal–Wallisrank sum test. If significant differences were found, the Mann Whitney’s U test wasconducted for comparison between the control and each dosage group. The incidenceof parental animals with clinical signs, and autopsy and histopathologicalfindings, the incidence of females with normal estrous cycles, incidence of weanlingswith histopathological findings, copulation, fertility and gestation index, neonatalsex ratio and completion rate of negative geotaxis were compared between the ASand control group using Fisher’s exact test. The incidence of pups with clinical signsor 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 bythe Wilcoxon rank sum test. The number of primordial follicles in the control andhighest dose groups was compared by Student’s t-test because the homogeneity ofvariance was indicated by the F-test. All of these statistical analyses were conductedusing the 5% level of probability as the criterion for significance.

Reproductive indices:

Reproductive performance of F0 and F1 parental animals:F0 generation:Copulation index (%)Fertility index (%)Delivery index (%)F1 generation:Copulation index (%)Fertility index (%)Delivery index (%)

Offspring viability indices:

Sex ratio, viability and body weight for F1 and F2 pups:F1 offspring:Sex ratio of pupsViability index of pups (%)Male pup weight during lactation (g)Female pup weight during lactation (g)F2 offspring:Sex ratio of pupsViability index of pups (%)Male pup weight during lactation (g)Female pup weight during lactation (g)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference was seen between control and substance-treated groups in the incidence of clinical signs of toxicity in either male or female F0 rats.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Water consumption and food consumption decreased partially during test period. 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):

Water consumption and food consumption decreased partially during test period. The body weight of F0 males and females was significantly lowered in the first 2 or 3 weeks of dosing at 3000 ppm.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No significant change

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: With increasing aluminium sulphate concentration water consumption decreases. This effect was taken into account for the results.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Adverse effects were not found in estrous cyclicity.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Adverse effects were not found in sperm parameters.

Reproductive performance:

no effects observed

Description (incidence and severity):

See table 1 attached

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

600 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed for any parameters determined in the study

2

Dose descriptor:

NOAEL

Effect level:

41 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed for any parameters determined in the study

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No significant effects

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant effects

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased body weight at PND 21 for 3000 ppm.

Sexual maturation:

no effects observed

Description (incidence and severity):

No significant effects

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Differences in absolute and relative organ weight for various organs: see table 3

Gross pathological findings:

no effects observed

Description (incidence and severity):

No significant effects

Histopathological findings:

no effects observed

Description (incidence and severity):

No significant effects

Effect levels (F1)

Remarks on result:

other: definitive conclusions could not be reached since paired-comparison data are not available to assess the effects of decreased water intake in the absence of exposure of Aluminium sulfate.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

In summary, Aluminium sulfate administered via drinking water at 120, 600 or 3000ppm

resulted in decreased water consumption. This change was associated with

decreased food consumption in the 600 and 3000ppm groups and decreased body

weight in the 3000ppm group. In the 3000ppm group, male and female pups had a

lower body weight on PND 21.

No definitive effects were found in the other reproductive/developmental parameters,

including developmental neurobehavioral toxicity.

Although there is a possibility that observed developmental effects are results

of decreased water consumption, more definitive conclusions could not be

reached since paired-comparison data are not available to assess the effects

of decreased water intake in the absence of exposure of Aluminium sulfate.

Applicant's summary and conclusion

Conclusions:

Aluminium sulfate administered via drinking water at 120, 600 or3000ppm resulted in decreased water consumption. This changewas associated with decreased food consumption in the 600 and3000ppm groups and decreased body weight in the 3000ppmgroup. In the 3000ppm group, male and female pups had alower body weight on PND(Post Natal Day) 21. At this dose, vaginal opening wasslightly delayed. No definitive effects were found in the otherreproductive/developmental parameters, including developmentalneurobehavioral toxicity. Although there is a possibility thatobserved developmental effects are results of decreased water consumption,more definitive conclusions could not be reached sincepaired-comparison data are not available to assess the effects ofdecreased water intake in the absence of exposure of Aluminium sulfate.NOAEL : 600 ppm = 41.0 mg/kg bw/day Aluminium sulfateCalculated for Aluminium potassium bis sulphate:NOAEL > 31 mg Aluminium potassium bis sulphate/kg bw/daybecause the NOAEL of second read across substance potassium sulfate is 1500 mg/kg bw/day.

Executive summary:

Aluminium sulfate administered via drinking water at

120, 600 or 3000ppm resulted in decreased water consumption.

This change was associated with decreased food consumption in the

600 and 3000ppm groups and decreased body weight in the 3000ppm group.

In the 3000ppm group, male and female pups had a lower body weight on PND(Post Natal Day) 21.

At this dose, vaginal opening was slightly delayed.

No definitive effects were found in the other reproductive/developmental parameters,

including developmental neurobehavioral toxicity.

Although there is a possibility that observed developmental effects are results

of decreased water consumption, more definitive conclusions could not be

reached since paired-comparison data are not available to assess the effects

of decreased water intake in the absence of exposure of Aluminium sulfate.

NOAEL : 600 ppm = 41.0 mg/kg bw/day Aluminium sulfate

Calculated for Aluminium potassium bis sulphate:

NOAEL > 31 mg Aluminium potassium bis sulphate/kg bw/day because the NOAEL

of second read across substance potassium sulfate is 1500 mg/kg bw/day.