Reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" can be considered as dermally non-absorbable as it is an inorganic, sparingly soluble ionic solid of lamellar structure. Particle size distribution of measured total dustiness (119.32 mg/g) was determined and the calculated MMAD and GSD values were used as input parameters for the prediction of the fractional deposition behaviour in the respiratory tract applying the Multiple Path Particle Dosimetry model (MPPD)(ver.2.11). Only very few amounts of the total dustiness fraction (4.3 %) will reach the pulmonary (alveolar) region. The vast majority of inhaled durst will be withheld in the naso-pharyngeal (head) region (89.1 %) and minor amounts in the tracheobronchial region (1.4 %). Deposits in the alveolar region would not get directly absorbed as the substance is a poorly soluble dust. The alveolar dust deposits would mainly be engulfed by alveolar macrophages. The macrophages will then either translocate particles to the ciliated airways or carry particles into the pulmonary interstitium and lymphoid tissues. Poorly water-soluble dusts depositing in the nasopharyngeal region could be coughed or sneezed out of the body or swallowed. Dusts depositing in the tracheo-bronchial region would mainly be cleared from the lungs by the mucocilliary mechanism and swallowed (c.f. ECHA TGD R7a, R.7.12.2.1).
Thus, direct inhalative systemic bioavailability is unlikely due to the deposition behaviour, crystalline structur and limited solubility. However, inhaled and subsequently swallowed amounts may decompose under the strong acidic conditions of the stomach and the released ions may become bioavailable. The assumption, that the substance acts, if at all, via its dissolved ions and therefore data on the ions can be used for hazard assessment, is in line with ECHA REACH TGD R6.

- As DNEL long-term exposure systemic inhalation worker, the German general dust limits for respirable dust (1.25 mg/m³) is used in compliance with ECHA REACH TGD R.8.
- Considering an exposure to 1.25 mg/m³ during the whole working week, the summed up exposure by use of standard default values (c.f. ECHA REACH TGD R.8: length of working day: 8h, working days/week: 5, inhalation volume: 10 m³ in 8 h, body weight: 70 kg) is 62.5 mg/worker/week or 0.89 mg/kg bw/week (12.5 mg/worker/day or 0.18 mg/kg bw/day).
- Highly conservative worst case assumption that inhaled dust is not cleared of the body by expectoration or sneezing, but completely cleared from the respiratory tract by the mucociliary mechanism and completely swallowed. Theoretically possible systemic absorption of small amounts via phagocytosis with subsequent transportation to the blood via the lymphatic system or engulfing by alveolar macrophages and subsequent transfer into the pulmonary interstitium and lymphoid tissues not considered for reason of simplification.
- Highly conservative worst case assumption that after swallowing, the material undergoes complete acidic dissolution in the stomach with 100 % release of the respective ions calcium, phosphonate and aluminium.
- Highly conservative worst case assumption that resorption rate of released ions is 100 %.
Calcium and phosphonates respectively phosphates are normal constituents of the body. As they are effectively processed and regulated in the body by natural physiological mechanisms, they are not expected to have any relevant toxic effect. For adults, an upper intake level "UL of 2500 mg of calcium per day for calcium intake from all sources" has been derived by the European Food Safety Authority (EFSA), who also stated, "that normal healthy individuals can tolerate phosphorus (phosphate) intakes up to at least 3000 mg/day without adverse systemic effects" (c.f. TOLERABLE UPPER INTAKE LEVELS FOR VITAMINS AND MINERALS, Scientific Committee on Food, Scientific Panel on Dietetic Products, Nutrition and Allergies, European Food Safety Authority (February 2006), http://www.efsa.europa.eu/fr/ndatopics/docs/ndatolerableuil.pdf). Thus, systemic intake of calcium and phosphonate deriving from exposure to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is even under highly conservative worst case considerations negligible and not expected to have any toxic effect.
Aluminium intake has been associated with some toxic effects to the nervous system and reproduction. A tolerated weekly intake (TWI) of 1 mg/kg bw/week has been derived by the European Food Safety Authority (EFSA) (c. f. Safety of aluminium from dietary intake, Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Food Contact Materials (AFC), The EFSA Journal (2008) 754, 1-34 http://www.efsa.europa.eu/de/efsajournal/doc/754.pdf and Scientific Option Re-evaluation of aluminium sulphates (E 520 -523) and sodium aluminium phosphate (E541) as food additives, The EFSA Journal (2018) 16(7):5372 https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2018.5372). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) increased the TWI from 1 mg/kg bw/week to 2 mg/kg bw/week (WHO (2012) Joint FAO/WHO Expert Committee on Food Additives. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series 65; http://whqlibdoc.who.int/publications/2012/9789241660655_eng.pdf). Considering that the total aluminium amount of inhaled substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (average aluminium content of the substance is 7.95 % by weight (7.70 – 8.20 %)) is bioavailable, the worker will be systemically exposed to approximately 0.07 mg/kg bw/week. However, beside the unlikelihood of 100 % clearance of inhaled dust via swallowing, according to EFSA (c.f. Statement of EFSA on the evaluation of a study related to the bioavailability of aluminium in food, The EFSA Journal (2011, 9(5) 2157; http://www.efsa.europa.eu/de/efsajournal/doc/2157.pdf), “oral bioavailability of aluminium from twelve different aluminium-containing compounds, including the food additives aluminium sulphate, … and sodium aluminium silicate, ranges merely from 0.02 to 0.21 %.” Considering this information, the real bioavailability of aluminium from inhaled and subsequent swallowed substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide", will be most likely significantly less than 1 %, which reduces the systemic exposure even under highly conservative worst case assumptions to less than 0.0007 mg/kg bw/week, a dose not expected to result in any toxic effect.
To underline that this dose level is highly unlikely to result in any toxic effects on reproduction, reprotoxicity data on soluble aluminium compounds with nontoxic counter moieties are read across. This read across is applicable considering that dissolved aluminium ions have similar bioaccessibility and bioavailability in biological fluids independently which aluminium compound has been dissolved.

This hypothesis is in line with with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.
The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The source substance and the target substance are considered to be similar as they are both:
(1) inorganic salts containing trivalent aluminium cations
(2) will ultimately dissociate into the common breakdown product Al3+ following chemical or biological hydrolysis and dissolution of the ionic bonds.
(3) do not have counter moieties which may have an own intrinsic toxicity

2. SOURCE AND TARGET CHEMICAL
Source substance aluminium sulphate is very soluble (>> 100g/L) and thus substantially more soluble than the target substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (0.68 g/L). The sulfur moiety of the source substance as well as the calcium and phosphate moieties of the target substance are not expected to have an own intrinsic toxicity. Sulfur is reduced or oxidized by organisms in a variety of forms. The element is present in proteins, sulfate esters of polysaccharides, steroids, phenols, and sulfur-containing coenzymes and is, as well as phosphate and calcium (see above), effectively processed and regulated in the body by natural physiological mechanisms.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance releases substantially higher aluminium amounts than the target. Therefore, the source substance is applicable to assess the intrinsic toxicity of this target substance moiety.

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)

Version / remarks:

Adopted on January 22, 2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

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:

obtained from Kanto Chemical Co., Inc. (Tokyo, Japan), Lot No. 007X1828 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:

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 (ion-exchanged water with and without the test substance), 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.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

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 aluminium sulfate 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:

P0 generation: Twenty-four F0 rats (5-week-old males and females)/sex/group were exposed to aluminium sulfate in drinking water at each dose level. After 10-week administration of aluminium sulfate, each female rat was mated with a male rat of the same dosage group until successful copulation occurred or the mating period of 2 weeks had elapsed. Administration of aluminium sulfate was continued throughout the mating.
P1 generation: 24 male and 24 female weanlings in each group were selected as P1 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 P0 parturition, and if the number of litters was insufficient, a second weanling pup in the litter was selected with care to prevent litter effects. For P1 matings, cohabitation of siblings was avoided. The day on which P1 parental animals were selected was designated as day 0 of dosing for the P1 generation.
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 aluminium sulfate 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. Aluminium sulfate contained in the drinking water for the control group was less than the quantitation limit (5 µg/mL).

Duration of treatment / exposure:

10 weeks before mating of P0 generation till post natal day 26 of F2 generation.
Administration of aluminium sulfate was continued throughout the mating, gestation and lactation periods.

Frequency of treatment:

daily

Details on study schedule:

Twenty-four P0 rats (5-week-old males and females)/sex/group were exposed to aluminium sulfate in drinking water at 0, 120, 600 or 3000 ppm. After 10-week administration of aluminium sulfate 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 aluminium sulfate was continued throughout the mating, gestation and lactation periods.
P0 parental male rats were necropsied after the parturition of paired females. P0 females were necropsied after weaning of their pups.
For the second (P1) generation, 24 male and 24 female weanlings in each group were selected as P1 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 P0 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 P1 parental animals were selected was designated as day 0 of dosing for the P1 generation. P1-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 P0 rats. Unselected F1 weanlings and all F2 weanlings were necropsied on PND 26.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

in ion-exchanged water (control group)

Doses / concentrations 2

Dose / conc.:

120 ppm (nominal)

Remarks:

in ion-exchanged water

Doses / concentrations 3

Dose / conc.:

600 ppm (nominal)

Remarks:

in ion-exchanged water

Doses / concentrations 4

Dose / conc.:

3 000 ppm (nominal)

Remarks:

in ion-exchanged water

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control P0 males, total ingested dose of aluminium from drinking water and food combined 1.62 mg aluminium/kg bw/day

Doses / concentrations 6

Dose / conc.:

8.6 mg/kg bw/day (actual dose received)

Remarks:

P0 males receiving 120 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 2.96 mg aluminium/kg bw/day

Doses / concentrations 7

Dose / conc.:

41 mg/kg bw/day (actual dose received)

Remarks:

P0 males receiving 600 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 8.06 mg aluminium/kg bw/day

Doses / concentrations 8

Dose / conc.:

188 mg/kg bw/day (actual dose received)

Remarks:

P0 males receiving 3000 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 31.2 mg aluminium/kg bw/day

Doses / concentrations 9

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control P0 females, total ingested dose of aluminium from drinking water and food combined 2.29 mg aluminium/kg bw/day

Doses / concentrations 10

Dose / conc.:

14.4 mg/kg bw/day (actual dose received)

Remarks:

P0 females receiving 120 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 4.50 mg aluminium/kg bw/day

Doses / concentrations 11

Dose / conc.:

71.5 mg/kg bw/day (actual dose received)

Remarks:

P0 females receiving 600 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 13.5 mg aluminium/kg bw/day

Doses / concentrations 12

Dose / conc.:

316 mg/kg bw/day (actual dose received)

Remarks:

P0 females receiving 3000 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 52.0 mg aluminium/kg bw/day

Doses / concentrations 13

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control P1 males, total ingested dose of aluminium from drinking water and food combined 1.93 mg aluminium/kg bw/day

Doses / concentrations 14

Dose / conc.:

10.7 mg/kg bw/day (actual dose received)

Remarks:

P1 males receiving 120 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 3.55 mg aluminium/kg bw/day

Doses / concentrations 15

Dose / conc.:

50.2 mg/kg bw/day (actual dose received)

Remarks:

P1 males receiving 600 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 9.78 mg aluminium/kg bw/day

Doses / concentrations 16

Dose / conc.:

232 mg/kg bw/day (actual dose received)

Remarks:

P1 males receiving 3000 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 38.5 mg aluminium/kg bw/day

Doses / concentrations 17

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control P1 females, total ingested dose of aluminium from drinking water and food combined 2.35 mg aluminium/kg bw/day

Doses / concentrations 18

Dose / conc.:

15.3 mg/kg bw/day (actual dose received)

Remarks:

P1 females receiving 120 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 4.72 mg aluminium/kg bw/day

Doses / concentrations 19

Dose / conc.:

74.2 mg/kg bw/day (actual dose received)

Remarks:

P1 females receiving 600 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 14.0 mg aluminium/kg bw/day

Doses / concentrations 20

Dose / conc.:

338 mg/kg bw/day (actual dose received)

Remarks:

P1 females receiving 3000 ppm in drinking water, total ingested dose of aluminium from drinking water and food combined 55.6 mg aluminium/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Prior to the present two-generation reproductive toxicity study, a dose-finding study was performed in male and female rats given drinking water containing aluminium sulfate at 0, 1000, 3000, 10,000 or 30,000 ppm.

Positive control:

no

Examinations

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 P0 and P1 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 was made 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 of AGD to 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 P1 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 P1 rats was recorded on the day of completion of these pubertal landmarks.
Spontaneous locomotor activity was measured at 4 weeks 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 [J.Genet Psychol 1940;56:439-45]. 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 then umber of errors were recorded. To prevent the exhaustion of the rats, no anima lwas allowed to remain in the water for more than 3 min 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 P1 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 5 µm 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.

Reproductive indices:

The following reproductive indices were calculated for the P0 and the P1 generation:
Copulation index (%) = (no. of animals with successful copulation/no. of animals paired)×10
Fertility index (%) = (no. of animals that impregnated a female or were pregnant/no. of animals with successful copulation)×100
Gestation index (%) = (no. of females that delivered live pups/no. of pregnant females)×100
Delivery index (%) = (no. of pups delivered/no. of implantations)×100

Offspring viability indices:

Viability indices were calculated for F1 and F2 offspring:
Viability index on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered)×100
Viability index on PND 4 (%) = (no. of live pups on PND 4/no. of live pups on PND 0)×100
Viability index on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull)×100

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 aluminium sulfate-treated groups in the incidence of clinical signs of toxicity in either male or female P0 rats.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the 600 ppm group, a subcutaneous mass was observed in the abdominal region of one P0 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 P0 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 P0 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 P0 females showed a significantly lower value during week 1 of dosing at 3000 ppm and during week 3 of lactation at 600 and 3000 ppm.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

In P0 males and females of all aluminium sulfate-treated groups, water consumption was significantly lower than in controls almost throughout the dosing period.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

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. There was no significant difference in the number of primordial follicles in the ovary of F1 females between control and 3000 ppm groups.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

During the premating period, aluminium sulfate produced no significant deviations in the estrous cycle of P0 and P1 females although a few control and aluminium sulfate-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 P0 generation, the absolute number of cauda epididymal sperm was significantly decreased at 3000 ppm (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 P1 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 aluminium sulfate-treated groups in either P0 or P1 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, 120 ppm and 3000 ppm groups and in one female of the control group in the P0 generation.
Among females with successful copulation, one female each in the control and 3000 ppm group and two females at 120 ppm in the P0 generation were not impregnated.
In addition, one pregnant P0 female each at 120, 600 and 3000 ppm 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 aluminium sulfate-treated groups in P0 generation.
No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other: However, it is very likely that all effects observed are no real toxicological effects. Most likely they are secondary effects determined by the decreased palatability of the drinking water in the aluminium sulfate-treated grougs.

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference was seen between control and aluminium sulfate-treated groups in the incidence of clinical signs of toxicity in either male or female P1 rats.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the 120 ppm group, one P1 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. At autopsy, various changes, including accumulation of ascitic and pleural fluid and dark purple discoloration of the liver and kidneys, were found.
One P1 male at 3000 ppm 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 P1 males and females exhibited no significant differences between the control and aluminium-sulfate-treated groups, except that P1 females of the 120 ppm 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 P1 males of the 600 and 3000 ppm groups, and during week 3 of lactation in P1 females of the same groups. There was also a transient significant increase in food consumption during week 6 of dosing in P1 females of the 120 ppm group.

Food efficiency:

not examined

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 600 ppm and 3000 ppm treated P1 males, and during weeks 3–6, week 8 and week 10 of dosing in 120 ppm treated males. In P1 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.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In P1 males, the absolute weights of the adrenals at 3000 ppm and the testes at 600 ppm 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 P1 female adults.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No dose-related gross lesions were found in P1 adults.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination of the reproductive organs revealed no compound-related alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

During the premating period, aluminium sulfate produced no significant deviations in the estrous cycle of P0 and P1 females although a few control and aluminium sulfate-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):

As for the sperm parameters examined for scheduled sacrificed adults, in P0 generation, the absolute number of cauda epididymal sperm was significantly decreased at 3000 ppm (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 P1 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 aluminium sulfate-treated groups in either P0 or P1 adults.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

In the P1 generation, one male in the control group, two males and one female in the 120 ppm group, one male in the 600 ppm group, and three males and one female in the 3000 ppm group did not copulate. Among females with successful copulation, two females each in the control, 600 ppm and 3000 ppm groups, and four females at 120 ppm in the P1 generation were not impregnated. In addition, one pregnant P1 female at 120 ppm 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 P1 generation.
No significant changes were observed in the number of implantations or pups delivered, and delivery index in either generation.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other: However, it is very likely that all effects observed are no real toxicological effects. Most likely they are secondary effects determined by the decreased palatability of the drinking water in the aluminium sulfate-treated grougs.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant changes were found in viability index.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 3000 ppm group, the body weight of male and female F1 pups was significantly lower than the control on PND 21.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

As for the sexual development of F1 male and female animals, vaginal opening was significantly delayed at 3000 ppm (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 aluminium sulfat-treated groups were noted in the age at preputial separation or body weight at the time of completion in males.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The AGD and AGD per cube root of the body weight ratio were not significantly different between control and aluminium sulfate-treated groups in male and female F1 pups.

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the 3000 ppm treated male and female F1 weanlings absolute and relative liver weights were significantly lower than the controls. Absolute spleen weight was also decreased significantly in both sexes of the 3000 ppm 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 3000 ppm group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 120 ppm group, but no significant difference was found in the incidence between the control and aluminium sulfate-treated groups.
External and internal gross observations revealed no compound-related alterations either in F1 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 weanlings.

Other effects:

no effects observed

Description (incidence and severity):

For the physical development of male and female F1 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 aluminium sulfate-treated groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

All male and female F1 pups in all groups achieved the 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 time of surface righting and negative geotaxis reflex.
At 4 weeks of age spontaneous locomotor activity at 10-min intervals and for 60 min was not significantly different between control and aluminium sulfate-treated 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.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

However, it is very likely that all effects observed are no real toxicological effects. Most likely they are secondary effects determined by the decreased palatability of the drinking water in the aluminium sulfate-treated grougs.

Results: F2 generation

General toxicity (F2)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant changes were found in viability index.

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 aluminium sulfate-treated groups during the preweaning period.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The AGD and AGD per cube root of the body weight ratio were not significantly different between control and aluminium sulfate-treated groups in male and female F2 pups.

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In F1 males, the absolute and relative weights of the thymus and spleen were significantly decreased in the 3000 ppm 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 change was a 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 600 ppm group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination of delivered pups revealed no malformed F2 pups in any groups.
External and internal gross observations revealed no compound-related alterations either in 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 F2 weanlings.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

For the physical development of 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 aluminium sulfate-treated groups.In female F2 pups, the completion rate of pinna unfolding on PND 2 was significantly lower in the 600 ppm 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.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In F2 pups, one female of the 600 ppm 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 600 ppm 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.

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

However, it is very likely that all effects observed are no real toxicological effects. Most likely they are secondary effects determined by the decreased palatability of the drinking water in the aluminium sulfate-treated grougs.

Applicant's summary and conclusion

Executive summary:

Aluminium sulfate administered via drinking water at 120, 600 or 3000 ppm resulted in decreased water consumption. This change was associated with decreased food consumption in the 600 and 3000 ppm groups and decreased body weight in the 3000 ppm group. In the 3000 ppm group, male and female pups had a lower body weight on PND 21. At this dose, vaginal opening was slightly delayed. No definitive effects were found in the other reproductive/developmental parameters, including developmental neurobehavioral toxicity.

A conservative evaluation of the data may led to the conclusion that the no observed adverse effect level of aluminium sulfate in this two-generation study is 600 ppm (41.0 mg/kg bw/day) for parental and offspring toxicity.

Since the dosing solution containing aluminium sulfate was pH 3.57–4.20, the acidity would decrease the palatability of drinking water in aluminium sulafte-treated groups. Decreased water consumption was associated with decreased food consumption by P0 and P1 males and females in the 600 and 3000 ppm groups and decreased body weight in P0 male and females in the 3000 ppm group. Since water deprived animals typically reduce their levels of feed consumption and consequently lower their body weight, decreased food consumption, body weight and absolut organ weights observed in the present study could be considered secondary to the decreased water consumption.

Considering these results, substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not expected to have an intrinsic toxicity to reproduction deriving from its aluminium moiety. As the further moieties - calcium and phosphonate - of the reaction mass do not have an intrinsic toxicity to reproduction either, substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" as such can be expected not to have an intrinsic toxicity to reproduction.