Aluminium tris(dihydrogen phosphate)

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008-2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

limited documentation, differences in pH of drinking water in control and test groups, no analysis of Al content in blood, urine and tissues.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance 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 enough to facilitate read-across for the following reasons:
(1) Both substances are inorganic salts containing a trivalent aluminium cation.
(2) Both substances will ultimately dissociate into the common breakdown products of the Al3+ following chemical or biological hydrolysis and dissolution of the ionic bonds.
In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

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)

Deviations:

yes

Remarks:

(limited information of methods, differences in the pH of drinking water in control and test groups)

GLP compliance:

yes

Limit test:

no

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

IUPAC Name:

aluminium sulphate

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 5 weeks
- Housing: Animals were housed individually, except for acclimation, mating and nursing periods, in suspended wire-mesh cages
From gestational day 17 (GD 17) to postnatal Day 21 (PND 21), individual dams and litters were reared in stainless-steel trays, and using wood chips as bedding (White Flake; Charles River Laboratories Japan, Inc., Yokohama, Japan).
- Diet: CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan, ad libitum (standard laboratory rodent chow).
- Al content in diet: 25 - 29 ppm (based on atomic absorption spectrometry)
- Water: deionized drinking water, ad libitum
- Al content in water: < 5 µg Al/mL (drinking water of controls)
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

ion-exchanged water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared freshly at least every 6 days and kept in a cool place until serving. Fresh drinking water was replaced at least once every 4 days.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks (until successful mating)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing, the first male was replaced by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individually except for nursing period
- For F1 mating, cohabitation of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of aluminium sulphate in drinking water were analysed in the first and last preparations and once every 3 months using high performance liquid chromatography (quantitation limit: 5 µg/mL) (analysed Al concentrations: 97.5 – 106.3% of the target).

Duration of treatment / exposure:

(P) Males: 10 weeks before mating.
(P) Females: 10 weeks before mating, during mating, gestation and lactation (weaning of pups: PND 26).
(F1) Males: starting at PND 21 - 25 (selection of F1 parental animals on PND 21- 25 to equalize mean body weights) until mating, during mating and production of F2 generation, until weaning (on PND 26)
(F1) Females: starting at PND 21 - 25 (selection of F1 parental animals on PND 21- 25 to equalize mean body weights) until mating, during mating, gestation and lactation until weaning (on PND 26)

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

Dams (P) delivered the offspring spontaneously and nursed their pups until PND 26. Litters were normalized on PND 4.
During PND 21 - 25, F1 parental animals were selected (desiganted as Day 0 or dosing) for the F1 generation. Exposure of the F1 weanlings started on the day of selection at the same doses as their parents.
F1-selected rats were necropsied in the same manner as described for F0 rats. The remaining F1 weanlings and all F2 weanlings were necropsied on PND 26.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 ppm (nominal)

Remarks:

in water

Doses / concentrations 2

Dose / conc.:

600 ppm (nominal)

Remarks:

in water

Doses / concentrations 3

Dose / conc.:

3 000 ppm (nominal)

Remarks:

in water

Doses / concentrations 4

Remarks:

Doses / Concentrations:
P males: 8.6, 41.0 and 188 mg Al2(SO4)3/kg bw/day; P females: 14.4, 71.5 and 316 mg Al2(SO4)3/kg bw/day
Basis:
other: mean dose value calculated based on reported body weight and water consumption (including aluminium from diet and drinking water)

Doses / concentrations 5

Remarks:

Doses / Concentrations:
P males: 2.96, 8.06 and 31.2 mg Al/kg bw/day; P females: 4.5, 13.5 and 52.0 mg Al/kg bw/day
Basis:
other: total doses of aluminium (food and water combined)

Doses / concentrations 6

Remarks:

Doses / Concentrations:
F1 males: 10.7, 50.2 and 232 mg Al2(SO4)3/kg bw/day; F1 females: 15.3, 74.2 and 338 mg Al2(SO4)3/kg bw/day
Basis:
other: mean dose value calculated based on reported body weight and water consumption (including aluminium from diet and drinking water)

Doses / concentrations 7

Remarks:

Doses / Concentrations:
F1 males: 3.55, 9.78 and 38.5 mg Al/kg bw/day; F1 females: 4.72, 14.0 and 55.6 mg Al/kg bw/day
Basis:
other: total doses of aluminium (food and water combined)

No. of animals per sex per dose:

24

Control animals:

other: concurent vehicle (total dose of aluminium via food and water: F0: 1.62 (males) and 2.29 (females) mg Al/kg bw/day; F1: 1.93 (males) and 2.35 (females) mg Al/kg bw/day)

Details on study design:

- Dose selection rationale: based on a range finding study (animals were orally exposed to 1000, 3000, 10,000 and 30,000 ppm aluminium sulphate for 7 weeks).

Results: Sacrifice of high-dose animals after 2 weeks due to water avoidance. Decreased water consumption was observed in the remaining groups. At 3000, 10,000 and 30,000 ppm, reduced food consumption and decreased body weights were observed. At necropsy, thickening of the limiting ridge in the stomach and atrophy of the thymus and spleen were observed at 10,000 ppm. Females exposed to 3000 or 10,000 ppm revealed decreased relative weights of the liver, thymus and spleen. Body weights of pups were decreased at 10,000 ppm on PND 4. Based on these results, 120, 600 and 3000 ppm were selected for the main study.

Examinations

Parental animals: Observations and examinations:

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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. Body weights of dams were recorded on GD 0, 7, 14 and 20 and PND 0, 4, 7, 14 and 21

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule for examinations: weekly. Food consumption of dams was recorded on GD 0, 7, 14 and 20 and PND 0, 7, 14 and 21

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: twice a week, and on GD 0, 4, 7, 11, 14, 17 and 20 and PND 0, 4, 7, 11, 14, 17, 19 and 21

Oestrous cyclicity (parental animals):

Estrous cyclicity was tested during the last 2 weeks of the premating and during mating (until evidence of copulation was detected based on daily vaginal lavage).
Repeated estrous cycles of 4 – 6 days were considered normal.

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
epididymis weight, sperm count in epididymides, sperm motility and sperm morphology

Litter observations:

STANDARDISATION OF LITTERS: Yes
Performed on day 4 postpartum: maximum of 8 pups/litter (4/sex/litter); no adjustment for litters with less than eight pups.

PARAMETERS EXAMINED:
F1 / F2 offspring: number of pups, sex ratio, live birth, postnatal mortality, presence of gross anomalies, weight gain, physical and behavioural abnormalities. Clinical signs of toxicity (daily) and body weight were measured on PND 0, 4, 7, 14 and 21.

GROSS EXAMINATION OF DEAD PUPS: Yes

DEVELOPMENTAL LANDMARKS
Developmental landmarks:
- Pinna unfolding in all F1 and F2 live pups (PND 1 to PND 4).
- Anogenital distance (AGD, measured using calipers on PND 4) in all F1 and F2 pups, normalized value of AGD to body weight, AGD/cube root of the body weight ratio
- Incisor eruption (one male and one female F1 and F2 pup/litter, evaluated on PND 8)
- Eye opening (starting on PND 12).
- Body weight (recorded on the day the criteria were fulfilled).

Neuromotor performance:
- Surface righting reflex, negative geotaxis and mid-air righting reflex (assessed on PND 5, 8 and 18, respectively), for one F1/2 male and female /litter).

Sexual maturation:
- Preputial separation (recorded daily in all F1 males selected as F1 parents, beginning on PND 35)
- Vaginal opening (recorded daily in all F1 females selected as F1 parents beginning on PND 25)
- Body weights (recorded on the day of completion of these pubertal landmarks).

NEUROBEHAVIOURAL EXAMINATIONS
Locomotor activity:
- Spontaneous locomotor activity (measured in 10 male and 10 female F1 rats which were randomly selected from each group at 4 weeks of age) using a multi-channel activity monitoring system (SUPERMEX; Muromachi Kikai Co., Ltd., Tokyo, Japan)


T-maze test:
- Water-filled multiple T-maze test (Biel (1940) (conducted in 10 male and 10 female F1 rats selected randomly from each group at 6 weeks of age)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: as soon as possible after parturition of their paired females
- Maternal animals: after weaning on PND 26.

GROSS NECROPSY
- External and Internal examination, including the cervical, thoracic, and abdominal viscera
- Organs examined: brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, seminal vesicles (with coagulating glands and their fluids), ventral prostate, uterus and ovaries
In 10 randomly selected F1 females from the control and highest dose groups, the number of primordial follicles was counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluations were performed:
- in all animals of the control and highest dose groups;
- in females showing abnormal estrous cycles, abnormal delivery or totally dead pups;
- in females and males without evidence of copulation or insemination;
- in all animals with grossly abnormal reproductive organs.
- Organs examined: testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina
When the highest dose group showed treatment-related changes in some tissues, examination of the same tissues was performed in the next lower dose group.

Postmortem examinations (offspring):

SACRIFICE
Following the adjustment of litter size on PND4, culled pups were euthanized and subjected to gross necropsy. Pups found dead before weaning were necropsied immediately. Not selected F1 pups as parental animals and all F2 pups were sacrificed on PND 26.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weights were determined for one male/female F1 and F2 weanling selected from each dam:
- brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries
- based on organ weight changes observed in the liver and spleen of the highest dose group in F1 and F2 generations, these tissues were histopathologically examined from 10 male and 10 female F1/F2 weanlings of the control and highest dose groups;
When the highest dose group showed treatment-related changes in some tissues, examination of the same tissues was performed in the next lower dose group.

Statistics:

Parametric data (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, behavioural test parameters, organ weight and sperm parameters) were analysed for homogeinity and distribution by Bartlett’s test.
Pre-weaning pups, body weight, AGD, viability, and age at the completion of developmental landmarks were similarly analysed using the litter as the experimental unit.
When homogeneity of distribution was established, One Way Analysis of Variance followed by Dunnett’s test were performed.
Data without homogeneity were analysed using the Kruskal–Wallis rank sum test followed by Mann Whitney’s U test.
The incidence of parental animals showing clinical signs, 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 analysed by Fisher’s exact test.
The Wilcoxon rank sum test was used to analyse the incidence of pups showing clinical signs and necropsy findings, completion rate of pinna unfolding, and the success rate of surface and mid-air righting reflex.
Student’s t-test was used to compare the number of primordial follicles in the control and highest dose group, homogeneity of variance was indicated by the F-test.
A 5% level of probability was used as criterion for significance.

Reproductive indices:

The following reproductive indices were calculated in F0 and F1 parental animals:
- Copulation index (%) (no. of animals with successful copulation/no. of animals paired) ×100;
- Precoital interval (days);
- Fertility index (%) (no. of males that impregnated a female or no. pregnant/no. of animals with successful copulation) × 100;
- Gestation index (%) (no. of females that delivered live pups/no. of pregnant females) × 100;
- Gestation length (days);
- Delivery index (%) (no. of pups delivered/no. of implantations) × 100;
- Estrous cycle in F0 and F1 females

Offspring viability indices:

The following parameters were analysed:

F1 and F2 offspring:
Maternal indices:
No. of litters;
No. of pups delivered;
Sex of all pups;
Sex ratio of pups total (no. of male pups/total no. of pups).

Viability index:
on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) × 100;
on PND 4 (%) = (no. of live pups on PND 4/no. of live pups on PND 0) × 100;
on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) × 100.

Individual body weight:
Male and female individual weight during lactation on PND 0, 4, 7, 14 and 21.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: significant reduction in body weight (P males/females in the first 2 -3 weeks of dosing); 600 and 3000 ppm: reduced food consumption

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: significant reduction in body weight (P males/females in the first 2 -3 weeks of dosing); 600 and 3000 ppm: reduced food consumption

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: 120, 600 and 3000 ppm: significant decrease in water consumption (P males and females)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

persistent diestrus (control and test animals, non-adverse)

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

3000 ppm: decreased number of cauda epdididymal sperm (non-adverse)

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical signs of toxicty were observed in the P generation. One unscheduled death was determined in the P mid-dose group at 2 weeks of gestation, the respective dam showed a subcutaneous mass in the abdominal region starting 5 weeks of dosing.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Significantly decreased body weights were observed in high-dose males and females in the first 2 - 3 weeks of dosing without showing a direct correlation to food consumption. Reduced food consumption was observed in mid- and high-dose animals at the following time points:
P mid-dose males - during the first week of dosing, high-dose males: during weeks 1, 8 and 13-14;
P mid-dose females: during week 3 of lactation, high-dose females: 1 week of dosing and during week 3 of lactation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant alterations in the estrous cycle were observed in P and F1 females during the premating period. However, persistent diestrus was determined in a few control and test animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
A significantly decreased number of cauda epididymal sperm (253.8 ± 61.3 × 1E+6/cauda compared to 286.3 ± 40.3 ×1E+6/cauda in test and control animals, respectively) was determined in high-dose males. However, after normalization to epididymal weight, the number of relative cauda epididymal sperm was comparable among the groups and hence, the effect is considered as non-adverse.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant differences on reproductive function or performance were observed including copulation, fertility, gestation index, precoital interval, gestation length, delivery index, number of implantations and number of litters or pups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Reduced liver (absolute and relative weight) and spleen (absolute weight) weights were determined in high-dose males. No alterations were observed in females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No dose-related gross lesions were found in any animal and dose group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No compound-related alterations were observed.

Effect levels (P0)

1

Dose descriptor:

other: LOAEL (systemic toxicity)

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Remarks:

(equivalent to 31.2 (males) and 52.0 (females) mg Al/kg bw/day)

Sex:

male/female

Basis for effect level:

other: decreased body weight gain; decreased food consumption

2

Dose descriptor:

other: NOAEL (systemic toxicity)

Effect level:

600 ppm (nominal)

Based on:

test mat.

Remarks:

(equivalent to 8.6 (males) and 13.5 (females) mg Al/kg bw/day)

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

3

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

>= 3 000 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 31.2 (males) and 52.0 (females) mg Al/kg bw/day, P generation

Sex:

male/female

Basis for effect level:

other: fertility - no adverse effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 generation: trauma in the perianal region and tail was observed in one control pup, hemimelia and oligodactyly were observed in one low-dose pup (non-adverse)

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 generation: decreased body weight in high-dose males and females on PND 21 and at scheduled sacrifice; F2 generation: decreased body weight in high-dose females on PND 21 and at sacrifice in both genders

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

F1 females, high-dose group: delayed vaginal opening

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 and F2 generation: liver, spleen, thymus, kidney, testes, epididymides, uterus, ovary and brain weights were affected by treatment

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
Viability was comparable on PND 0, 4 and 21 amomg the groups in any generation (F1 and F2).

CLINICAL SIGNS (OFFSPRING)
F1 generation
No signs indicative for clinical toxicity were observed in test animals. The observed trauma in the perianal region and tail or hemimelia and oligodactyly is not considered as adverse due to the low incidence (observed in 1 control and 1 low-dose pup, respectively).

F2 generation
No signs indicative for clinical signs were observed in any dose group.

BODY WEIGHT (OFFSPRING)
F1 generation
Significantly reduced body weights of high-dose animals (males and females) compared to the control were observed on PND 21 and at sacrifice (for details, please refer to Tables 1 and 2).
F2 generation
Reduced body weight was determined in high-dose females on PND 21 and at sacrifice in both genders (for details, please refer to Tables 1 and 2).


SEXUAL MATURATION (OFFSPRING)
F1 generation
Significantly delayed vaginal opening was observed in high-dose females (31.4 ± 1.7 compared to 29.5 ± 2.1 days in control) correlating to a slightly increased body weight compared to controls (119.0 ± 13.3 versus 109.6 ± 11.6 g).
No significant differences were noted regarding the age of preputial separation. No changes in body weights at the time of preputial completion were noted.


ORGAN WEIGHTS (OFFSPRING)
F1 generation
Significant decreases in liver (absolute and relative organ weight), spleen, thymus, kidney, testes and epididymides weights (absolute weights) and increased relative brain weights were observed in high-dose animals. The absolute uterus weight was decreased in high-dose animals.

F2 generation
Weights of the thymus, spleen (absoulte and relative weights), liver and epididymides (absolute) were significantly reduced. Relative brain weight was increased in high-dose males. Reduced absoulte brain weight was observed in females of the mid-dose group (please refer to Tables 1 and 2).

GROSS PATHOLOGY (OFFSPRING)
No alterations were found in either the F1 or F2 generation during the preweaning period (data not shown).

HISTOPATHOLOGY (OFFSPRING)
No dose-related histopathological changes were determined in the liver or spleen of male and female F1 and F2 weanlings.

OTHER FINDINGS (OFFSPRING)
SEX RATIO
The sex ratio was comparable among all test groups in the F1 or F2 generation.

PHYSICAL DEVELOPMENT
In the F1 generation and F2 males of all test groups, completion rate of pinna unfolding, age at completion of incisor eruption and eye opening were comparable among the groups. F2 females revealed a significantly lower completion rate of pinna unfolding on PND 2 compared to controls in the mid-dose group (17.0 ± 35.4%, compared with 45.8 ± 46.9% in mid-dose females and controls, respectively). Otherwise, females of the F2 generation did not show significant differences in regard to the measured developmental landmarks.

AGD and AGD per cube root of body weight ratio were comparable among the groups in the F1 and F2 generation.

NEUROMOTOR DEVELOPMENT
The development of reflexes (surface righting reflex on PND5, negative geotaxis reflex on PND8 and midair righting reflex on PND 18) was comparable among the groups in any generation. Further, the response times of surface righting and negative geotaxis reflexes were not affected. However, 1 mid-dose F2 female did not achieve the mid-air righting reflex on PND 18 in 1/3 trials without affecting the mean success rate between the control and mid-dose group (100 ± 0.0% versus 98.4 ± 7.3%).

RESULTS ON PARENTAL ANIMALS OF THE F1 GENERATION
CLINICAL SIGNS AND MORTALITY
No clinical signs of toxicity were observed in the parents selected from the F1 generation.
Incidental death occured in the low- and high-dose group: 1 low-dose male died at 9 weeks of dosing. Prior to death, soiling of periocular and perinasal fur and decreased locomotor activity were determined. Necropsy revealed ascitic, accumulation of pleural fluid and dark purple discoloration of the liver and kidneys. Furthermore, 1 high-dose male died at week 12 of dosing without showing clinical signs of toxicity.

WATER CONSUMPTION
Statistically reduced water consumption was determined in the F1 generation in all dose groups: Low-dose males showed significantly decreased water consumption (evident during 3-6, 8, 10 weeks of dosing). Water consumption of mid-and high-dose animals was decreased throughout the dosing period. Decreased water consumption was determined in F1 females of the low-dose group (evident during 9 - 10 weeks of dosing), the mid-dose group (evident during week 10 of dosing and week 3 of lactation) and the high-dose group (evident throughout the entire dosing period).

FOOD CONSUMPTION
Significantly reduced food consumption was determined in the mid- and high-dose group either in week 10 of dosing (males) or week 3 of lactation (females). Moreover, reduced food consumption was observed in low-dose females during week 6 of dosing.

BODY WEIGHT
Body weights were comparable among the groups except for F1 low-dose females which revealed an increased body weight during week 6 - 8 compared to controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cycles were comparable in F1 females among the groups although some control and test animals had persistent diestrus.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects on the evaluated sperm parameters were determined (the number of cauda epididymal sperm, number of testis sperm, percentage of motile sperm and progressively motile sperm, swimming speed and pattern and percentage of morphologically abnormal sperm).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance was comparable among the groups: copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations, number of litters or pups delivered were not affected by treatment.

ORGAN WEIGHTS
Absolute organ weight of adrenals was significantly decreased in adult F1 males of the high-dose group compared to control animals. Mid-dose males showed significantly reduced absolute testis weight.
Organ weights of females of the F1 generation did not differ from controls.

PATHOLOGY
No dose-related gross lesions were observed in the F1 generation. Reproductive organs revealed no compound-related alterations in adults of the F1 generation.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

fertility

Generation:

F1

Effect level:

>= 3 000 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 38.5 (males) and 55.6 (females) mg Al/kg bw/day, F1 generation

Sex:

male/female

Basis for effect level:

viability

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 38.5 (males) and 55.6 (females) mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

sexual maturation

body weight and weight gain

organ weights and organ / body weight ratios

3

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 9.78 (males) and 14.0 (females) mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The bodyweight of female F2 pups were lower than controls on PND 21 (3,000 ppm). No sinificant differences in bodyweight of male F2 pups between control and AS treated groups were noted.

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver, spleen, thymus, kidney, testes, epididymides, uterus, ovary and brain weights were affected by treatment.

In 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 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

Effect levels (F2)

1

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 38.5 (males) and 55.6 (females) mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

600 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 9.78 (males) and 14.0 (females) mg Al/kg bw/day

Sex:

male/female

Basis for effect level:

other: no adverse effects noted

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1.Organ weights of F1 and F2 male weanlings

 

	Control		120 ppm		600 ppm		3000 ppm
	F1	F2	F1	F2	F1	F2	F1	F2
Number of animals	22	21	20	18	22	22	22	21
Body weight (%)	100%	100%	NS	NS	NS		87.44**	90.31**
Brain
Absolute weight (%)	100%	100%	NS	NS	NS	NS	NS	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	113.22**	112.11**
Thymus
Absolute weight (%)	100%	100%	NS		NS	NS	81.33**	79.84**
Relative weight (%) (g/100 g bw)	100%	100%	NS	89.29*	NS	NS	NS	87.92**
Liver
Absolute weight (%)	100%	100%	NS	NS	NS	NS	80.60**	87.78**
Relative weight (%)(g/100 g bw)	100%	100%	NS	NS	NS	NS	91.61**	NS
Kidney^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	89.62**	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Spleen
Absolute weight (%)	100%	100%	NS	NS	NS	NS	76.40**	80.43**
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	86.93**	88.36**
Testis^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	90.44*	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Epididymis^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	88.02**	93.62*
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS

NS: no statistically significant differences were observed

**: significantly different from the control, p<0.01;

*: significantly different from the control, p<0.05;

^a: total weights of the organs on both sides.

Table 2. Organ weights of F1 and F2 female weanlings

 

	Control		120		600		3000
	F1	F2	F1	F2	F1	F2	F1	F2
Number of animals	22	22	20	18	22	21	21	21
Body weight (%)	100%	100%	NS		NS		89.91**	91.34**
Brain
Absolute weight (%)	100%	100%	NS	NS	NS	102.5*	NS	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS		110.20**	110.05**
Thymus
Absolute weight (%)	100%	100%	NS	NS	NS	NS	81.72**	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Liver
Absolute weight (%)	100%	100%	NS	NS	NS	NS	84.80**	86.24**
Relative weight %) (g/100 g bw)	100%	100%	NS	NS	NS	NS	94.26*	94.56**
Kidney^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	NS	NS
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Spleen
Absolute weight (%)	100%	100%	NS	NS	NS	NS	86.65*	84.11**
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Ovary^a
Absolute weight (%)	100%	100%	NS	NS	NS	NS	NS	84.52**
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS
Uterus
Absolute weight (%)	100%	100%	NS	NS	83.85*	NS	78.47**	81.49*
Relative weight (%) (g/100 g bw)	100%	100%	NS	NS	NS	NS	NS	NS

NS: no statistically significant differences were observed

**: significantly different from the control, p<0.01;

*: significantly different from the control, p<0.05;

^a: total weights of the organs on both sides.

Applicant's summary and conclusion

Conclusions:

Interpretation of the results of this study is complicated by the effect of aluminium sulphate treatment on fluid consumption. It is noted that high concentrations of aluminium sulphate led to a reduction in the pH of the drinking water which may have reduced the palatability. In addition food consumption of F0 and F1 females was also reduced relative to the controls during week 3 of lactation.

Therefore reductions in food and water consumption during lactation mean that observed effects in the F1 and F2 generations (such as reduced organ weights) may be secondary effects rather than direct effects of aluminium sulphate consumption. Therefore, the utility of this data as stand-alone data is limited.