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Effects on fertility

Description of key information

Reproductive toxicity (OECD 422,oral, rat): NOAEL >= 1000 mg/kg bw/day as aluminium chloride (equivalent to 180 mg Al/kg bw/day and 567 mg Al oxide/kg bw/day)

Link to relevant study records

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Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 September 2006-3 November 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
: Temporary deviations from the maximum level of relative humidity occurred; acclimatization period was shorter due to the late delivery of animals; Age of animals was 9 and 11 weeks instead of recommended 12 weeks
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
STEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males (9 weeks), females (11 weeks)
- Weight at study initiation: in body weight were ± 20% of the sex mean
- Fasting period before study:
- Housing:
Pre-mating period: 5 animals/sex/Macrolon plastic cage
Mating period: female and male (1:1) in Macrolon plastic cages
Post-mating period: 5 males/ Macrolon plastic cage; females - individually in Macrolon plastic cage
Lactation PND-1-4: Offspring were kept with dams until termination
General care: sterilized sawdust was used as bedding material and paper was supplied as cage enrichment material
- Diet: ad libitum to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany). Each batch was analyzed for nutrients and contaminants on a regular basis (frequency is not reported).
- Water: tap water ad libitum
- Acclimation period: 4 days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3.0°C (actual range 19.9 - 23.4°C)
- Humidity (%):30 - 70% (actual range 37 - 95%)
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours’ darkness per day

ADDITIONAL INFORMATION
- Identification (F0) – earmark and tattoo.
- Randomization was performed by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.


Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared within 4 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance. No adjustment was made for specific gravity of the vehicle and formulation.

Rationale for vehicle: selection on vehicle based on information provided by the sponsor (Report, 2007, p.13).

Details on mating procedure:
- M/F ratio per cage: 1:1 (during breeding); One female was mated with one male from the same treatment group.
- Proof of pregnancy: Detection of sperm in the vaginal lavage or intra-vaginal copulatory plug was considered as postcoitum day 0.
- After mating was confirmed, the male and female were separated.
- The mating period continued for 14 days. After this time, any females who did not show evidence of mating were separated from their males (Report, 2007, p.14).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of aluminium levels observed in the formulations of the test substance in Milli-U water showed that the formulations were close to the target concentrations i.e. 94-103%.

Duration of treatment / exposure:
Males were exposed for 28 days – 2 weeks prior to mating, during mating and up to termination/adequate
Females were exposed for 37 - 53 days – 2 weeks prior to mating, during mating, during post-mating, and during 3 days of lactation.

Administered volume – 5 ml/kg bw. Actual doses were adjusted to the latest body weight.
Frequency of treatment:
Daily, 7 days per week. Administration was performed approximately at the same time each day with a maximum of 4 hours difference between the earliest and latest dose.

Animals were dosed up to the day prior to scheduled necroscopy.
Details on study schedule:
- Only parental animals F0 were mated.
- Males were 9 weeks at mating.
- Females were 11 weeks at mating.
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
Al chloride; corresponding to 7.2 mg Al/kg bw/day assuming the substance is 18% Al by mass
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Al chloride; corresponding to 36 mg Al/kg bw/day assuming the substance is 18% Al by mass
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Al chloride; corresponding to 180 mg Al/kg bw/day assuming the substance is 18% Al by mass
No. of animals per sex per dose:
F0 animals – 40 animals per sex and 20 rats (10 males and 10 females) per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
“Dose levels were based on the results of the dose range finding study (NOTOX project 473524) and were set in consultation with the sponsor” (Report, 2007, p.15).

Animals (5 males and 5 females) were randomly selected for functional observations, clinical laboratory investigations, macroscopic examination and organ weight determination.

Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded.

Analysis of bedding, paper, diet and water did not reveal any findings that might affect the study outcomes.
Positive control:
Not required.
Parental animals: Observations and examinations:
Mortality – twice daily.

Clinical signs – once daily for all animals. Once prior to start of treatment and at weekly intervals outside the home cage in a standard arena during the study. Arena observations were not performed when the animals were mating or housed individually.
All clinical symptoms, the time of onset, degree and duration were recorded and graded based on 1-4 grade (fixed scale) scores:
- Maximum grade 1: grade 0- absent; grade 1 – present.
- Maximum grade 3 or 4: grade 1-slight, grade 2 – moderate, grade 3 – severe, and grade 4 – very severe.

Cage debris of pregnant females were examined for evidence of abortion or premature birth, signs of difficult or prolonged parturition were recorded.

Functional observation
Males (n=5) on week 4,
Females (n=5) – during lactation:

- hearing ability;
- papillary reflex;
- static righting reflex
- grip strength
- motor activity test (recording period: 12 hours during overnight for individual animals, using computerized monitoring system).


Body weight (males and females): on the first day of exposure and then weekly. Females were examined on GD 0, 4, 7, 11, 14, 17 and 20 and on lactation days (LD) 1 and 4.

Food consumption (males and females): weekly but not recorded during mating period. Following evidence of mating, food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and PND 1 and 4.

Water consumption: no quantitative data available as no effect was suspected.

Clinical examination
Animals (5 males and 5 females selected randomly from each group) were fasted with a maximum 20 hours overnight before blood sampling but water was provided. Blood samples were drawn from the retro-orbital sinus and collected with EDTA for hematological parameters (0.5 ml), with citrate for clotting tests (0.9 ml) and Li-heparin for clinical biochemistry parameters (0.5 mL).

Hematology: red blood cells (RBC); red blood cell distribution width (RDW); white blood cell (WBC) count; differentiation of white blood cells; reticulocytes; hematocrit; haemoglobin; mean corpuscular haemoglobin concentration; mean corpuscular volume; haematocrit; platelet count.

Coagulation Potential: prothrombin time (PT); activated partial thromboplastin time (APTT).

Clinical biochemistry: alanine aminotransferase; aspartate amonotransferase; alkaline phosphatase; total protein; albumin; total bilirubin; urea; creatinine; glucose; cholesterol; sodium; potassium; chloride; calcium; inorganic phosphate.

Macroscopic examination: the cranial, thoracic and abdominal tissues and organs were examined with special attention to reproduction organs.
The numbers of implantation sites and corpora lutea were recorded from all females.

Reproductive parameters recorded: male number paired with; mating date; confirmation of pregnancy; delivery day.
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Effect of Al test compound on spermatogenic cycle in males was not examined.
However, histopathological examination of testes and epididymides was performed (information below).
Litter observations:
Number of live and dead pups at the first litter check (first day of lactation) and daily.

Clinical signs (pain, distress or discomfort) were recorded and animals with clinical signs that were non-transient in nature were sacrificed for humane reasons (based on OECD ENV/JM/MONO/2000/7).

The individual weight of all live pups on PND 1 and 4 of lactation.

Sex of all pups on days 1 and 4 of lactation (by assessing ano-genital distance).

The number of pups with physical or behavior abnormalities daily.

No litter standardization was performed as the pups were killed on day 4.
Postmortem examinations (parental animals):
Male animals were killed on day 29 of study.

Female animals were killed at day 4 post-partum or shortly after.

All parental animals were macroscopically examined for internal/external abnormalities, including cervical, thoracic and abdominal viscera examination with special attention to the reproductive organs.

For 5 animals from each group and sex, the body weight and weights of the adrenal gland, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus were recorded. From all remaining animals/sex/group the epididymides and testes weights only were recorded.

The tissue from selected rats (n=5/10 per group) of both the control and high dosage groups were examined, in addition, any abnormalities from the lower dose groups were examined too.
Tissues and organs histopathologically examined (5 surviving animals/sex/group): adrenal glands; aorta; brain (cerebellum, mid-brain, cortex); caecum; cervix; clitoral gland; colon; coagulation gland; duodenum; epididymides; heart; ileum; jejunum; kidneys; liver; lungs- infused with formalin; lymph nodes- mandibular, mesenteric; esophagus; ovaries; pancreas; Peyer’s patches (jejunum, ileum); pituitary gland; preputial gland; prostate gland; rectum; sciatic nerve; seminal vesicles; spinal cord- cervical, mid-thoracic, lumbar; spleen; sternum with bone marrow; stomach glandular and keratinized; testes*; thymus; thyroid including parathyroid; trachea; urinary bladder; uterus; vagina; all gross lesions/abnormalities. From all remaining animals: cervix; clitoral gland; coagulation gland; epididymides*; ovaries; preputial gland; prostate gland; seminal vesicles; testes*; uterus; vagina; all gross lesions.

Histopathological examination

PAS sections of the testes and epididymides were examined for staging in accordance with the guidelines published in the Society of Toxicologic Pathology Position paper (Lanning et al., 2002) with particular focus on the presence of retained spermatids, missing germ cell layers, multinucleate giant cells and sloughing of spermatogenic cells.

In addition, all zones of the epididymides were evaluated for leukocyte infiltration, sperm granuloma, change in prevalence of cell types, change in constitutive cells, aberrant cell types in the lumen and phagocytosis of sperm (Appendix 6, p.7).

The following tissues were examined from animals suspected of infertility:

• male (n=4): coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles, and testes and
• female (n=3) - the cervix, clitoral gland, ovaries, uterus, and vagina.

Tissues/organs taht were not examined microscopically: eyes with optic nerve and Harderian gland; female mammary gland area; femur including joint; larynx; salivary glands; skeletal muscle; skin; lacrimal gland, exorbital; nasopharynx; tongue, “as no signs of toxicity of target organ involvement were indicated”
Postmortem examinations (offspring):
All offspring were killed on day 4 of lactation.
All offspring were sexed and externally examined (no details provided). The stomach was examined for the presence of milk.
Defects or cause of death were evaluated.
The brain of 1 pup /sex/litter was collected and fixed in neutral phosphate buffered 4% formaldehyde solution (Klinipath, Duiven, The Netherlands).
The terminal body weight and brain weight was recorded from 1 pup/sex/litter.
Statistics:
The following statistical methods were used to analyze the data:

• If the variables follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on pooled variance estimate was used to compare the treatment groups with the control. Analyses were done separately by sex

• The Student’s t-test (Sokal, 1981) to compare pup organ weight.

• The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data was not assumed to follow normal distribution.

• The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

• All tests were two-sided and in all cases p<0.05 was accepted asstatistically significant.

No statistical analysis was performed on histopathology findings. LABCAT software version HP4.33 was used for report preparation.

Test statistics were calculated on the basis of exact values for means and pooled variances.
Reproductive indices:
The following reproductive indices for each exposed group were calculated:

- percentage mating (number of females mated x100/number of females paired);

- fertility index (number of pregnant femalesx100/number of females paired);

- conception rate (number of pregnant femalesx100/number of females mated );

- gestation index (number of females bearing live pupsx100/number of pregnant females);

- duration of gestation (number of days between confirmation of mating and the beginning of parturition);

- percentage of live males at first litter check (number of live male pups at first litter check x100/number of live pups at first litter check);

- percentage of live females at first litter check (number of live female pups at first litter check x100/number of live pups at first litter check);

- percentage of postnatal loss days 0 to 4 postpartum (number of dead pups on day 4 postpartumx100/number of live pups at first litter check).
Offspring viability indices:
Viability index (number of live pups on day 4 postpartum/number of live pups at first litter check x 100).

The individual weights of all live pups on days 1 and 4 of lactation were measured and the sex of all pups determined by measuring the ano-genital distance.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No critical clinical signs of toxicity were reported in either sexes over observation period.
Detail: Bilateral alopecia of various body parts (head, neck, forelegs) was observed in 1 female exposed to 40 mg/kg Al chloride basic, however, similar dermal abnormalities within the same magnitude were observed in 2 females from control group.
Increased salivation and excretion were noted in both females and males exposed to 1000 mg/kg of Al chloride basic.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males
1000, 200 and 40 mg/kg
No effects on body weight and body weight gain were observed in Al treated males compared to control animals.
No changes in food consumption were observed in Al treated males compared to control animals.

Females
1000 mg/kg
Body weight was statistically significantly lower compared to the control group during:
• pre-mating period*/week 2, day 8:
240.0 ± 12.0 vs 254 ± 12.4 in control group (< 5.5%);

• first day of mating* : 254 ± 11.3 vs 271 ± 13.1 in control group (< 6.3%);

• at the beginning of gestation, days 0, 4 and 7:
256 ± 12.9* vs 274 ± 13.1 in control group (< 6.6%);
274 ± 9.9** vs 295 ± 14.9 in control group (<7.2%)
287 ± 13.0** vs 308 ± 16.8 in control group (6.9%).

However, body weight in pregnant females recovered from gestational day 11 (311 ± 14.8 vs. 329 ± 15.4 in control group) until the end of the study (up to lactation day 4).

Body weight gain
Body weight gain was also significantly decreased at the beginning of treatment – the pre-mating period** , day 8, week 2:
1 ± 2.1 vs. 6 ± 3.4 in control group but significantly increased at the end of gestation, day 20*:
67 ± 8.7 vs. 58 ± 5.3 in the control group (> 13.5%).

No changes in body weight gain were observed by the end of study (lactation day 4).

No effects were observed on body weight and body weight gain were observed in Al treated females at doses 200 and 40 mg/kg compared to the control animals.

Absolute and relative food consumption
Females
Mean absolute food consumption (g/animal/day) of females exposed to 1000 mg/kg of Al chloride basic were lower by 24% during the 1-2 weeks of treatment/pre-mating phase (16 ± 1.1 compared to 21 ± 0.1 in control group. Relative food consumption was also lower by 17.3% during the same period (67 ± 3.4 mg/kg /day compared to 81 ± 1.3 mg/kg/ day in control group).

No effects were observed on absolute and relative food consumption in Al treated females during the pre-mating, mating and 2 weeks of post-mating period at doses 200 and 40 mg/kg compared to control animals.
Increased relative food consumption during days 14 - 17 and 17 - 20 of the post-coitum/gestational period was observed, but the increases were not statistically significant.

* - Dunnett t-test based on pooled variance significant at 5%)
**- Dunnett t-test based on pooled variance significant at 1%)


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Al compound was administered with drinking water by gavage at doses 0, 40, 200 and 1000 mg/kg Al chloride basic. Formulations of the test substance exhibited unbound aluminium concentrations consistent with actual concentrations 94, 102 and 103% of the target formulation concentrations of 8, 40 and 200 mg/mL.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Histopathological studies were performed on testes and epididymides.
Authors reported that “there were no treatment-related effects on the spermatocytic cycle”, Appendix 6, p.9) and “the assessment of the integrity of the spermatogenic cycle did not provide any evidence of impaired spermatogenesis”.

However, no other details were provided on the results of performed examination of PAS sections of testes and epididymides.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance

Females
Pre-coital time:

Control group(n=10)
10/10 animals were mated during first 3 days. Mean pre-coital time – 1.9 days (n=10).

1000 mg/kg(n=10)
8/10 animals (80%) were mated during first 3 days, 2 animals (20%) – during 4th day of mating. Mean pre-coital time – 2.6 days.

200 mg/kg(n=10)
8/10 animals (80%) were mated during first 3 days, 2 animals (20%) – during 4th day of mating. Mean pre-coital time -2.7 days.

40 mg/kg(n=9)
5 animals were mated during first 3 days of mating, during 4 day - 2 females, 8 day -1 female, and 12 day -1female. Mean pre-coital time – 4.4 days.

The delay in mating did not show a relationship with dose of the test substance and was not reported as statistically significant.

Three Al treated females (2/40 mg/kg group; 1/200 mg/kg group) and 1 female from control group were suspected of infertility (no specific details provided).

Males
No data on mating performance was provided for males, however, 3 animals were suspected to be infertile (no details provided).

Reproduction data

Females
1000 mg/kg
The studied reproduction parameters were unaffected in 10/10 animals. One female experienced complete litter loss, but this is unlikely to be related to treatment.

200 mg/kg
1 female from was not pregnant after the mating period.

40 mg/kg
1 female from was non-pregnant after mating period.

Overall, there were no treatment-related effects on reproduction parameters.

Mating performance, duration of gestation, number of corpora lutea, number of implantation sites, and number of dead and living pups at first litter check were also similar between the control and treated groups.

Breeding parameters
The breeding parameters studied were unaffected by Al treatment.

Postnatal loss between PND 0 - 4 and viability index were similar for the control and Al treated groups.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Males
1000, 200, 40 mg/kg
No toxicologically significant changes in absolute and relative organ weight between Al treated groups and control animals.

Females
1000 mg/kg
Statistically significant decrease in the absolute brain weight (by 6.8%) was observed in females from the high dose group compared to control animals
- 1.92 ± 0.05 compared to 2.06 ± 0.11 in control group, respectively;

No Al treatment-related differences were observed in the relative (organ/body weight ratio,%) brain weight between Al treated and control animals.

200 mg/kg
Statistically significant decrease in the absolute kidney weight (by 15%) was observed in the 200 mg/kg dosed animals compared to control:
- 1.94 ± 0.07 vs. 2.28 ± 0.23 in control group, respectively;

No Al treatment-related differences were observed in the relative kidney weight between Al treated and control animals.


GROSS PATHOLOGY (PARENTAL ANIMALS)
Males
1000 mg/kg

Stomach
Red foci were observed in the glandular mucosa of the stomach of 5/10 animals associated with thickening of the glandular mucosa or limiting ridge in 2 of these 5 animals.

No other treatment-related macroscopic changes were observed in males:

1000 mg/kg
Pelvic dilatation of kidneys was observed in 1 animal.

200 mg/kg
No Al treatment macroscopic effects were observed in 10 from 10 examined animals.

40 mg/kg
From 10 animals, 7 animals were without pathological finding.
Enlarged mesenteric and mandibular lymph nodes were observed in 3 of 10 animals. In 1 of them, red discoloration of mesenteric lymph nodes was also noted.
A tan focus on the right lateral lobe of the liver was observed in 1 animal/10.
Kidney cist 2x2 was observed in 1 animal (N16) from 10 examined.

Control group
A reduced size of the left thigh muscle was reported in 1 /10 animals.

Females
1000 mg/kg
No Al treatment macroscopic effects were observed in 10/10 examined animals.

200 mg/kg
Red foci in thymus in 1 animal from 10 examined.

40 mg/kg
Fluid in uterus was detected in 1/10 animals. Alopecia in animals N53.

Control group
Red foci in thymus (1/10) and alopecia (1/10) were observed in control females. Enlarged liver and spleen in 1 (number 47) animal.


HISTOPATHOLOGY (PARENTAL ANIMALS)
Maternal/Paternal toxicity

Males
1000 mg/kg
A minimal, mild or moderate subacute inflammation of the glandular stomach mucosa and minimal to moderate superficial mucosal eosinophilic spheroids were present in all examined animals.

Females
1000 mg/kg
A minimal, mild or moderate subacute inflammation of the glandular stomach mucosa and minimal to moderate superficial mucosal eosinophilic spheroids were present in all examined animals.

Authors stated that all other microscopic findings were in the range of background pathology existing in Wistar rats of this age and strain and occurred at similar incidence and severity in both control and treated rats.


Histopathological findings on suspected infertility
Female
The following histopathological findings were reported for 4 female (1/control group; 2 dosed with 40 mg/kg; 1 dosed with 200mg/kg) microscopically examined due to suspected infertility:

Control group
Uterine horn decidual thickening (indicating previous pregnancy) and moderate local inflammatory hemorraghic ulceration (suggestive of a separated placentation);

40 mg/kg
Uterine horn decidual thickening indicating previous pregnancy (1 animal), uterine dilatation and trilaminar vaginal epithelium presence (1 animal, indicator of active oestrus cycle);

200 mg/kg
No indication of past or present reproductive activity.

Males
No histopathological abnormalities were detected in the reproductive organs of males suspected of infertility (1/control group; 2/40 mg/kg; 1/200mg/kg).

Emergency killing
1 control female was killed in extremis but did not show any lesions which could be considered as a cause of mortality.


OTHER FINDINGS (PARENTAL ANIMALS)
Neurobehavioral performance

Males, Females

1000, 200 and 40 mg/kg
No Al treatment effects on hearing ability, papillary reflex, static righting reflex and grip strength during the functional observations in both males and females were noted.
No treatment-related effects on motor activity among Al exposed females and males compared to control groups were recorded, however, high variability in motor activity data was evident.

Hematology

Males(n = 5)

1000 mg/kg
Statistically significant changes were observed in males at the end of treatment period:
- decreased hemoglobin level* (9.6 ± 0.2 vs 9.9 ± 0.1 in control group, < 3%),
- decreased mean corpuscular hemoglobin concentration* ( 20.99 ± 0.18 vs 21.42 ± 0.18 in control group, by 2%) ;
- increased platelets content* (1234 ± 180 vs. 972 ± 143 in control group, by 21%);

200 mg/kg
Statistically significant decreased Hb level in males compared to control animals:
- decreased hemoglobin level** (9.5 ± 0.2 vs 9.9 ± 0.1 in control group, by 4%)

40 mg/kg
Statistically significant decrease in white blood cells compared to control animals –
- decreased white blood cells 7.8 ± 1.1 vs. 11.4 ± 1.9 in control group by 32%
- decreased hemoglobin level* (9.6 ± 0.2 vs 9.9 ± 0.1 in control group, < 3%)

Females (n=5)
1000 mg/kg
- Statistically significant decrease in mean corpuscular hemoglobin concentration* ( 20.17 ± 0.42 vs 20.86 ± 0.49 in control group, by 3.3%) .

40 mg/kg
- Statistically significant increased white blood cells* compared to control animals (8.1 ± 1.1 vs. 5.4 ± 1.6, respectively, by 50%);


Clinical biochemistry (blood serum)

Males (n = 5)
1000 mg/kg
Statistically significant changes were observed in males at the end of treatment period:
- decreased in alkaline phosphatase* ( 96 ± 4 vs 125 ± 23 in control group, respectively, by 23.2%);
- decreased total protein* (54.8 ± 2.0 vs. 59.0 ± 2.6 in control group, respectively, by 7.2%);
- decreased albumin* ( 29.3 ± 0.9 vs. 31.1 ± 1.2 in control group, respectively, by 5.7%)
- increased potassium** (4.24 ± 0.09 vs. 3.83 ± 0.23 in control group, respectively, by 10.7%);
- increased inorg. phosphate levels*( 2.78 ±0.05 vs. 2.43 ± 0.31, by 14.4%)

200 mg/kg
- increased potassium* (4.16 ± 0.14 vs. 3.83 ± 0.23 in control group, respectively, by 8.6%).

Females (n = 5)
1000, 200, 40 mg/kg
No Al treatment effects on studied clinical biochemistry parameters were observed in any group compared to control.


* - Dunnett t-test based on pooled variance significant at 5%)
** - Dunnett t-test based on pooled variance significant at 1%)
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
200 mg/kg bw (total dose)
Based on:
test mat.
Remarks:
corresponding to 36 mg Al/kg bw/day
Sex:
male/female
Basis for effect level:
gross pathology
Dose descriptor:
LOAEL
Remarks:
local effects
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Remarks:
corresponding to 180 mg Al/kg bw/day
Sex:
male/female
Basis for effect level:
gross pathology
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Remarks:
corresponding to 180 mg Al/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose tested
Critical effects observed:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
No Al treatment related effects on viability were observed.

CLINICAL SIGNS (OFFSPRING)
During in- life litter check, no milk in the stomach, cold, weak and/or pale pups’ appearance and scabs on the tail were observed. The authors state that these findings were of small appearance, within the normal biological variation for rats of this age and strain and were not associated with Al treatment.
The pups of female No.56 (40 mg/kg) showed signs of cannibalism.

BODY WEIGHT (OFFSPRING)
No changes in body weight were observed for male and female pups born from the Al-treated dams and control dams.

SEXUAL MATURATION (OFFSPRING)
Not examined.

ORGAN WEIGHTS (OFFSPRING)
Only absolute/relative brain weights were studied.

Males
No changes in the absolute brain weight for male pups born from the Al-treated dams and control dams were observed.

Females
1000 mg/kg bw
Statistically higher absolute brain weight (by 7%) was noted in female pups born from dams treated to 1000 mg/kg Al chloride basic.
- 0.568±0.0328 vs. 0.530± 0.0445 in control group, respectively.

No changes in the relative brain weight (organ to body weight, %) for the 1000 mg/kg dose group of pups were observed.

40 and 200 mg/kg bw
No differences in the absolute and relative brain weight were observed between female pups born from dams exposed to 200 and 40 mg/kg of the Al chloride basic and female pups born from control females.


GROSS PATHOLOGY (OFFSPRING)
Please see section “clinical signs”.

HISTOPATHOLOGY (OFFSPRING)
Not performed.

OTHER FINDINGS (OFFSPRING)
Postnatal development

No toxicologically significant changes in pups development were noted during PND 0-4 (no further details were provided).
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
corresponding to 180 mg Al/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose tested
Reproductive effects observed:
not specified
Conclusions:
With focus on rationale for reliability:

This study involved short-term and sub-chronic exposure and observed no adverse reproductive and developmental effects. Aluminium was administered by a relevant route (oral) at multiple dose levels before mating and at a critical period of embryo- , organogenesis and development.

No adverse effects on reproductive behavior, mating criteria and histological structure of examined reproductive organs in males and females of rats exposed to aluminium chloride (basic) (gavage) at doses of 40, 200 and 1000 mg aluminium chloride basic/kg during pre-mating, gestation and short-time lactation period were reported. Suggested NOAEL for reproductive toxicity (lack of reproductive /breeding, mating impairment and early postnatal developmental effects) of 1000 mg/kg.
The study conformed to an international screening test guideline, was performed according to Good Laboratory Practice and therefore contributes to the weight of evidence for the absence of reproductive, breeding and mating activity impairment due to sub-acute exposure of rats exposed to 40 mg aluminium chloride basic/kg, 200 mg/kg and 1000 mg/kg. Klimisch Score of 2 (reliable with accepted deviations) is considered appropriate.
Executive summary:

This GLP study was performed in accordance with OECD Test Guideline (TG) 422 and adds to the weight of evidence for the absence of reproductive/breeding, mating impairment and early postnatal developmental effects due to short-term exposure to high doses of aluminium chloride (basic).

No mortality or clinical signs of intoxication were observed in male and female Wistar rats due to treatment with Al chloride basic at dose levels of 40, 200, and 1000 mg/kg body weight.

Treatment with Al chloride basic by oral gavage revealed paternal toxicity (irritation effect on glandular stomach mucosa, local effect) at 1000 mg/kg in both the male and female Wistar rats. Based on findings observed macroscopically (red foci or thickening of the grandular mucosa of the stomach) and supported by microscopic examination, the maternal/parental No Observed Adverse Effect Level (NOAEL) for local toxic effects on stomach was established at 200 mg/kg and LOAEL – at level 1000 mg/kg, for both males and females.

Several statistically significant changes in clinical biochemistry parameters were observed at 1000 mg/kg suggesting a possible impact on the blood system (decreased Hb level in males, MCHC in both Al treated males and females), on the liver (decreased total protein and albumin in blood serum) and possibly the kidney functions (increase potassium level) at this dose. Decreased Hb levels were observed in two other doses in males but no dose response relationship was observed. Lack of relevant base line values for the observed clinical data limit the interpretation of the results. The authors consider the and clinical biochemistry and haematology changes observed at 1000 mg/kg to be of slight nature and generally within the range expected for rats of this age and strain. Because any morphological correlates were absent, these changes were considered not indicative of organ dysfunction and not of toxicological significance.

No reproduction, breeding and early post-natal developmental toxicity was observed in rats at 1000 mg/kg body weight for males and females. Based on the reported results, a NOAEL for reproduction, breeding and early post-natal developmental toxicity was suggested at a level of 1000 mg/kg bw.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (10-fold interval between doses used in this study, urinanalysis was not perfromed).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
10-fold interval between doses in this study, urinanalysis was not performed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles river Laboratories Japan, Inc.
- Age at study initiation: (P) 5 wks
- Housing: animals were housed individually, except for the acclimation, mating and nursing periods, in metal-bracket-type cages with wire-mesh floor
- Diet: standard rat diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum, Al content in diet, analyzed by flame atomic absorption spectrometry for each lot of diet, ranged from 22 ppm to 29 ppm
- Water: deonized drinking water with (treatment group) or without (control group) dose concentrations, water given to controls contained < 5 µg Al/mL
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: drinking water
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: fresh dosing solutions were prepared at least once every 7 days and the drinking water was replaced at least once every 5 days
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until successful copulation occured or the mating period of 2 weeks had elapsed
- Proof of pregnancy: vaginal smears were examined daily for presence of sperm, and the presence of sperm in the vaginal smear and/or a vaginal plug were considered as evidence of successful mating, detection of sperm in the vaginal lavage was designated as day 0 of gestation
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged: from day 17 of gestation to day 21 after delivery, dams and litters were reared using wood chips as bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
concentrations of AAS in drinking water were analyzed in the first and last preparations and once every 3 months, ans verified using high performance liquid chromatography (the quantitation limit -5 µg/mL)
Duration of treatment / exposure:
F0 males: 10 weeks prior to mating, during mating and up to termination after the parturition of paired females
F0 females: 10 weeks prior to mating, during mating and during lactation period until sacrifice after weaning of their pups (PND26)
F1: selected at PND21-25, exposure occured at the same doses and schedule as their parents
Frequency of treatment:
7 days/week
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
- Age at mating of the mated animals in the study: 13-15 weeks
Dose / conc.:
50 ppm (nominal)
Remarks:
For actual doses received see table1 under any other information on materials and methods including tables.
Dose / conc.:
500 ppm (nominal)
Remarks:
For actual doses received see table1 under any other information on materials and methods including tables.
Dose / conc.:
5 000 ppm (nominal)
Remarks:
For actual doses received see table1 under any other information on materials and methods including tables.
No. of animals per sex per dose:
parental and F1: 24 males and females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a dose-range finding study
- Rationale for animal assignment: by stratified random sampling based on body weight
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all F0 and F1 parental rats were observed at least twice a day for general appearance and behavior. Dams were checked 3 times daily on days 21-25 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through the study. For dams, body weight was recorded weekly until evidence of copulation was detected and then on gestational days 0, 7, 14 and 20 and days 0, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined once a week and mean daily diet consumption calculated as g food/ day: Yes
Time schedule for examinations: weekly. For dams, food consumption was recorded through the exposure period, except during cohabitation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice a week, through the exposure period, except during cohabitation.
Oestrous cyclicity (parental animals):
Daily vaginal lavage samples were evaluated for each female for estrous cyclicity throughout the last 2 weeks before the cohabitation period and during cohabitation until evidence of copulation was detected. The presence of sperm in the vaginal smear and/ or a vaginal plug was considered as evidence of successful mating, and the day of successful mating was designated as day 0 of gestation.
Sperm parameters (parental animals):
Sperm parameters were determined in all F0 and F1 adult males on the day of sacrifice; The right testis was used to count testicular homogenization-resistant spermatid heads using a hemacytometer; The right epididymal cauda was weighed and used for sperm analysis (sperm number, sperm motility and morphology; Caudal sperm numbers were enumerated using a hemacytometer under a light microscope; For sperm motility (percentage of motile sperm and progressively motile sperm, swimming speed and pattern) were determined using a computer-assisted cell motion analyzer (TOX IVOS; Hamilton Thorne Bioscience, Beverly, MA, USA); Sperm morphology was evaluated for 200 sperm (stained with eosin and mounted on a slide glass) per male under a light microscope.
Litter observations:
STANDARDISATION OF LITTERS:
Performed on day 4 postpartum: Yes; maximum of 8 pups /litter (4/sex/litter), randomly selected.

PARAMETERS EXAMINED:
The following parameters were examined in all pups from F0 and F2 parents (F1 and F2 litters): number of pups, sex of pups, live birth, stillborn members per litter, and gross anomalies Clinical signs of toxicity (daily) and the body weight of live pups were measured on PND 0, 4, 7, 14 and 21.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

OTHER:
Developmental landmarks:
- Pinna unfolding in all F1 and F2 live pups (for from PND 1 to PND 4); Eye opening beginning on PND12;
- The anogenital distance (AGD) was measured on PND 4 in all F1 and F2 pups using calipers, and the normalized value of AGD to body weight, AGD/cube root of the body weight ratio, was calculated (Gallavan et al., 1999);
- Incisor eruption beginning in one male and one female F1 and F2 pup selected from each dam were evaluated on PND 8 and eye opening beginning on PND 12, and continued until each pup achieved the criteria;
- The body weight of the respective F1 pups was recorded on the day the criteria were fulfilled.

Neuromotor performance:
- For the same F1 and F2 pups, surface righting reflex, negative geotaxis and mid-air righting reflex were assessed on PND 5, 8 and 18, respectively.

Sexual maturation:
- Preputial separation were observed daily for male in all F1 offspring selected as F1 parents beginning on PND 35 and
- Female vaginal opening were observed daily for female in all F1 offspring selected as F1 parents beginning on PND 25 until completion.
- The body weight of the respective F1 rats was recorded on the day of completion of these pubertal landmarks.

NEUROBEHAVIORAL EXAMINATIONS:
Locomotor activity:
- Spontaneous locomotor activity was measured in 10 male and 10 female F1 rats randomly selected from each group at 4weeks of age. A multi-channel activity monitoring system (SUPERMEX; Muromachi Kikai Co., Ltd., Tokyo, Japan) was employed.
Animals were placed individually in transparent polycarbonate cages [285(W)x 450(D) x 210(H) mm, CL-0108-1; CLEA Japan Inc., Tokyo, Japan], and spontaneous motor activity was measured using SUPERMEX (Muromachi Kikai Co., Ltd., Tokyo, Japan), which was placed above the cage to detect changes in heat across multiple zones of the cage with an infra-red sensor. Spontaneous motor activity was determined at 10-min intervals and for a total 60 min.

Spatial learning ability (T-maze test):
- Spatial learning ability was conducted using water-filled multiple T-maze test (Biel’s type) in 10 male and 10 female F1 rats selected from each group at 6 weeks of age.
The water temperature of the maze was maintained at 21.0–22° C. Each rat was allowed to swim three times in a straight channel on the day before the trial, and then tested in the maze with three trials per day for the following three consecutive days. The time required to reach the goal and the number of errors were recorded. To prevent the exhaustion, no animal was allowed to remain in the water for more than 3 min in any trial.
Postmortem examinations (parental animals):
SACRIFICE
- F0 and F1 parental male animals: after a parturition of their paired females;
- Maternal animals: were evaluated for estrous cycle stage by examination of the vaginal smear after weaning of pups, and euthanized in the proestrus stage by exsanguination under ether anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic, and abdominal viscera;
- The number of uterine implantation sites was recorded for each female;
- The testis and epididymis in males were prepared for microscopic examination and weighed;
- The brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididmydes, seminal vesicles (with coagulating glands and their fluids), ventral prostate, uterus and ovaries in males and females were weighed before fixation, fixed and underwent macroscopic examination; thyroid and seminal vesicles were weighed after fixation;
- 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 number of primordial follicles in the right ovary was counted for 10 F1 females randomly selected from the control and highest dose groups. Every 20th section was mounted on a slide and stained with hematoxylin–eosin. About 40 sections per ovary were used to determine the primordial follicles.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathological evaluations were performed in F0 and F1 animals of the control and highest dose groups.

Of these animals, the testes, epididymides, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina were embedded in paraffin ,sectioned, stained with hematoxylin–eosin and examined under a light microscope.
Postmortem examinations (offspring):
SACRIFICE
Non-parental F1 weanlings and all F2 offspring were euthanized under ether anesthesia at PND 26 of age.

HISTOPATHOLOGY / ORGAN WEIGTHS
For one male and one female F1 and F2 weanlings selected from each dam:
- the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries were removed, weighted and prepared for microscopic examination;
-Since test substance-related organ weight changes were found in the thymus, liver and spleen weight and in the liver and spleen weight of females in the highest dose group in F1 and F2 generations, they were histopathologically examined for the randomly selected 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups. Paraffin sections were routinely prepared, stained with hematoxyllin-eosin and examined using a light microscope.
Statistics:
Bartlett’s test for homogeneity of variances (p<0.05) was applied for homogeneity of distribution for parametric data (body weight, food and water consumption, length of estrous cycle and gestation, precoital interval, number of implantations and pups born, delivery index, reflex response time, age at sexual maturation, parameters of behavioral tests, organ weight and sperm parameters);
For preweaning pups, body weight, AGD, viability, and age at completion of developmental landmarks were similarly analyzed using litter as experimental unit.
One way analysis of variance (p<0.10) was performed when homogeneity of distribution was established.
If a significant difference was detected, Dunnett’s test was conducted for comparisons between control and individual treatment groups (p<0.01 or 0.05).
Data without homogeneity were analyzed using Kruskal–Wallis rank sum test (p<0.10). If significant differences were found, Mann Whitney’s U test was conducted for comparison between control and each treatment group (p<0.01 or 0.05). Fisher’s exact test (p<0.01 or 0.05) was used to compare incidence of parental animals with clinical signs, and autopsy and histopathological findings, 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 between AS and control group.
Wilcoxon rank sum test (p<0.01 or 0.05) was used to analyzed incidence of pups with clinical signs or autopsy findings per litter, completion rate of pinna unfolding in each litter, and success rate of surface and mid-air righting reflex.
Student’s t-test (p<0.01 or 0.05) was used to compare number of primordial follicles in control and highest dose groups because homogeneity of variance was indicated by F-test.
All of these statistical analyses were conducted using 5% level of probability as criterion for significance.
Reproductive indices:
The following reproductive indices for each exposed group were calculated in F0 and F1 generation parental animals:
- Copulation index (%) for males and females(no. of animals with successful copulation/no. of animals paired)×100;
- Precoital interval(days);
- Fertility index(%) for males and females (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;
- Gestation length (days);
- Delivery index(%)(no. of pups delivered/no. of implantations)×100;
- Estrous cycle in F0 and F1 females.
Offspring viability indices:
For F1 and F2 offspring
Maternal indices:
No of litters;
No of pups delivered;
Sex ratio of pups total (no. of male pups/total no. of pups).

Viability index was calculated:
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.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
some effects in different live stages observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
some effects in different live stages observed
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: details provided in table1
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
during premating period a few AAS treated animals had persistent diestrous (not adverse)
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
P0 males and females (50, 500 and 5000 ppm)
No treatment-related parental deaths or clinical signs of intoxication at any treatment groups in either male or female P0 rats.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
WATER CONSUMPTION
P0 males (50, 500 and 5000 ppm)
- was significantly lower during the entire 14-week treatment period in all three dose groups
P0 females (50 ppm)
- was significantly lower than in controls during premating period (weeks 1,9 and 10 of dosing), 1 week of gestation and week 1 of lactation);
(500 ppm)
- was significantly lower than in controls during premating, gestation and lactation period;
(5000 ppm)
- was significantly lower than in controls during premating, gestation and lactation period.

FOOD CONSUMPTION
P0 males (5000 ppm)
- was decreased in the 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
P0 females (500 ppm)
- significantly decreased during week 1 of premating period.
(5000 ppm)
- significantly decreased at 1 week of premating period, and 2-3 weeks of lactation period.

BODY WEIGHT
P0 males (5000 ppm)
- significantly decreased in the first 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
P0 females (5000 ppm)
- significantly decreased in the first 1 week of premating period and at the end of lactation period (21 day).
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test compound was administered with drinking water. The mean aluminium ammonium sulfate and elemental Al intakes during the whole dosing period in P0 males, P0 females, P1 males and P1 females provided in Table 1 (any other information on materials and methods including tables).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
P0 females (50, 500 and 5000 ppm)
estrous cycle
- during a premating period, a few AAS-treated P0 rats had persistent diestrus, however:
- no significant changes in the incidence of P0 females with normal estrous cycle (4-5 days) compared to the control animals were observed during the premating period;
- no significant differences in the estrous cycle between AAS treated and control groups were noted.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
P0 males (50, 500 and 5000 ppm)
There were no significant differences in the number of testis sperm and cauda epidymal 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 AAS-treated groups in P0 adults (however, no details were provided on the results of performed examination).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance
P0 animals
The authors reported that some animals failed to copulate, impregnate or deliver live pups, however:
- no significant differences were observed between the control and AAS-treated groups in P0 generation (24 animals per group) for 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. Overall, there were no treatment-related effects on reproduction parameters.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P0
- relative kidney weight was significantly increased in P0 females (500 and 5000 ppm);
- absolute weight of the pituitary glands was significantly decreased in P0 females (5000 ppm)

GROSS PATHOLOGY (PARENTAL ANIMALS)
P0 males and females
No AAS treatment- related gross lesions were observed in either generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P0 males and females
No treatment related histopathological changes of the reproductive organs were observed.
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 36.3 and 59.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 5.35 and 8.81 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 36.3 and 59.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Critical effects observed:
not specified
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
P1 males and females (50, 500 and 5000 ppm)
No treatment-related parental deaths or clinical signs of intoxication at any treatment groups in either male or female P1 rats.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
WATER CONSUMPTION
P1 males (50 ppm)
- no significant changes were found compared to the control animals.
(500 and 5000 ppm)
- significantly lower than in controls throughout the treatment period
P1 females (50 ppm)
- was significantly lower than in controls during week 4 and 8-10 weeks of premating period;
(500, 5000 ppm)
- was significantly lower than in controls during premating, gestation and lactation period;

FOOD CONSUMPTION
P1 females (5000 ppm)
- significantly decreased at 2-3 weeks of lactation period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
P1 males (50, 500 and 5000 ppm)
- no significant changes were observed in the Al treated and control groups.

BODY WEIGHT
P1 males (5000 ppm)
- significantly decreased in the first 1 week of premating period.
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.
P1 females (5000 ppm)
- significantly decreased in the first 1-2 weeks of premating period;
(50, 500 ppm)
- no significant changes were observed in the Al treated and control groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test compound was administered with drinking water. The mean aluminium ammonium sulfate and elemental Al intakes during the whole dosing period in P0 males, P0 females, P1 males and P1 females provided in Table 1 (any other information on materials and methods including tables).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
P1 females (50, 500 and 5000 ppm)
estrous cycle
- during a premating period, a few AAS-treated P1 rats had persistent diestrus, however:
- no significant changes in the incidence of P1 females with normal estrous cycle (4-5 days) compared to the control animals were observed during the premating period;
- no significant differences in the estrous cycle between AAS treated and control groups were noted.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
P1 males (50, 500 and 5000 ppm)
There were no significant differences in the number of testis sperm and cauda epidymal 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 AAS-treated groups in P1 adults (however, no details were provided on the results of performed examination).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance
P1 parental animals
The authors reported that some animals failed to copulate, impregnate or deliver live pups, however:
- no significant differences were observed between the control and AAS-treated groups in P1 generation (24 animals per group) for 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. Overall, there were no treatment-related effects on reproduction parameters.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Adults (P1)
- absolute weight of the pituitary glands was significantly decreased in P1 males and females (5000 ppm);
- absolute thymus decreased significantly in P1 females (5000 ppm);
- relative weight of seminal vesicle was significantly decreased in P1 males (50 ppm);
- absolute brain weight was significantly decreased in P1 females (500 ppm).

GROSS PATHOLOGY (PARENTAL ANIMALS)
P1 males and females
No AAS treatment- related gross lesions were observed in either generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P1 males and females
No treatment related histopathological changes of the reproductive organs were observed.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 ppm
Based on:
test mat.
Remarks:
AS; equivalent to 6.57 and 9.36 mg Al/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 44.2 and 61.1 mg Al/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 44.2 and 61.1 mg Al/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Critical effects observed:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
bodyweight for F1 males and females (5000 ppm) decreased significantly, for F2 (5000ppm) lower body weight but not significantly
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
significantly delayed vaginal opening (F1 - 5000ppm)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see table3 + 4
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
F1 generation (50, 500 and 5000 ppm)
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation

CLINICAL SIGNS (OFFSPRING)
F1 generation
Clinical (external gross) examination during the preweaning period revealed microphthalmia, a rudimentary tail, trauma and scab on right hindlimb and crushing of incisor/malocclusion in a few F1 pups in control and AAS-treated groups (no data provided); however, there no significant differences in incidence between the control and AAS-treated animals were observed (data not shown);

BODY WEIGHT (OFFSPRING)
F1 generation (5000ppm group, F1 males and F1 females)
- body weight of male and female pups was significantly lower on PND 21 and on PND 14 and 21, respectively, compared to the control pups.

SEXUAL MATURATION (OFFSPRING)
F1 females (5000 ppm)
vaginal opening
- was significantly delayed (32.3±1.8 days of age, compared to 30.2±2.1days of age in control). Body weight at the time of vaginal opening was slightly higher than the control (122.0±15.7 g. versus 115.8±12.6 g.) but no statistically significant difference was found.
F1 males (50, 500 and 5000 ppm)
the age at preputial separation
- no significant differences between control and AAS-treated groups were found and no changes were found in body weight at the time of completion of separation.

ORGAN WEIGHTS (OFFSPRING)
F1 males (Table 2) (5000 ppm)
Body weight was significantly decreased (85.97% compared to the control group); relative brain weight was significantly increased; absolute kidney weight was significantly decreased but the relative kidney weight was significantly increased ; absolute and relative thymus weight was significantly decreased;
absolute and relative liver weight was significantly decreased; absolute and relative spleen weight was significantly decreased; absolute weight of the adrenal glands was decreased significantly; absolute weight of testis and epididymis was significantly decreased.

F1 females (Table 3) (5000 ppm)
Body weight was significantly decreased (87.59% compared to the control group); relative brain weight was significantly increased; relative kidney weight was significantly increased; absolute thymus weight was significantly decreased; absolute liver weight was significantly decreased; absolute and relative spleen weight was significantly decreased; absolute weight of the adrenal glands was decreased significantly; absolute weight of uterus was significantly decreased.

F1 females (Table 3) (500 ppm)
weight of the adrenal glands was decreased significantly.

F1 females (5000ppm)
Number of primordial follicles in the ovary was no different between Al treated and control females (data not shown).

GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations
F1 males and females
Gross observations did not reveal any compound-related lesions in F1 weanlings or in pups found dead during the lactation period (no data provided that any pups were found dead during the lactation period).

HISTOPATHOLOGY (OFFSPRING)
F1 males and females
No dose-related histopathological changes in the liver and spleen of male and female F1 weanlings and of the thymus in males in both generations.

OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT LANDMARKS
PHYSICAL DEVELOPMENT
F1 males/females (50, 500 and 5000 ppm)
- Completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening , the AGD and AGD per cube root of the body weight ratio were not significantly different between the control and AAS-treated groups (data not shown).

NEUROMOTOR DEVELOPMENT
F1 males/females (50, 500 and 5000 ppm)
- No significant changes were observed in the achieved day of the surface righting reflex (PND5), negative geotaxis reflex (PND8) and midair righting reflex (PND 18).
- No significant changes were observed in the response time of surface righting and negative geotaxis reflex (data not shown).

BEHAVIOR PERFORMANCE
SPONTANEOUS LOCOMOTOR ACTIVITY
F1 males (50, 500 and 5000 ppm)
- was not significantly different between control and AAS treated males at 10-min intervals and for 60 min;
F1 females (500 ppm)
Spontaneous locomotor activity was significantly decreased during the 40-50 and 50-60 minutes after start of test but no significant differences were observed in total activity for 60 min; no changes in spontaneous locomotor activity for 10 min intervals or for a total of 60 min between the control and the other AAS-treated groups in females

T MAZE TEST
Learning and memory performance in T-maze test (pre-test swimming trials in the straight channel)
F1 males and females (50, 500 and 5000 ppm)
- no significant changes were observed in the elapsed time to traverse the straight channel.
- no significant changes were observed in the elapsed time and number of errors on days 2–4.
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 36.3 and 59.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Generation:
F1
Effect level:
5.41 mg/kg bw/day
Based on:
test mat.
Remarks:
Al
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 36.3 and 59.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
female
Basis for effect level:
sexual maturation
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
500 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 5.35 and 8.81 mg Al/kg bw/day in P0 males and females, respectively
Sex:
female
Basis for effect level:
sexual maturation
Critical effects observed:
not specified
VIABILITY (OFFSPRING)
F2 generation (50, 500 and 5000 ppm)
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation

CLINICAL SIGNS (OFFSPRING)
F2 generation
No gross abnormalities in F2 pups were found in any groups.

BODY WEIGHT (OFFSPRING)
F2 generation (5000ppm, F2 males and females)
- body weights were lower than controls around time of weaning but no statistically significant differences were found.

SEXUAL MATURATION (OFFSPRING)
No effects observed.

ORGAN WEIGHTS (OFFSPRING)
F2 males (Table 2) (5000 ppm)
Body weight was significantly decreased (92.21% compared to the control group); relative brain weight was significantly increased; absolute and relative thymus weight was significantly decreased; absolute liver weight was significantly decreased; relative kidney weight was significantly increased; absolute and relative spleen weight was significantly decreased.

F2 females (Table 3) (5000ppm)
Body weight was significantly decreased (90.50% compared to the control group); relative brain weight was significantly increased; absolute thymus weight was significantly decreased; absolute and relative liver weight was significantly decreased; relative kidney weight was significantly increased; absolute and relative spleen weight was significantly decreased; relative weight of the adrenal glands was increased significantly; absolute ovary weight was significantly decreased; absolute uterus weight was significantly decreased.

GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations
F2 males and females
Gross observations did not reveal any compound-related lesions in F2 weanlings or in pups found dead during the lactation period (no data provided that any pups were found dead during the lactation period).

HISTOPATHOLOGY (OFFSPRING)
F2 males and females
No dose-related histopathological changes in the liver and spleen of male and female F2 weanlings and of the thymus in males in both generations.

OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT LANDMARKS
PHYSICAL DEVELOPMENT
F2 males/females (50, 500 and 5000 ppm)
- Completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening , the AGD and AGD per cube root of the body weight ratio were not significantly different between the control and AAS-treated groups (data not shown).

NEUROMOTOR DEVELOPMENT
F2 males/females (50, 500 and 5000 ppm)
- No significant changes were observed in the achieved day of the surface righting reflex (PND5), negative geotaxis reflex (PND8) and midair righting reflex (PND 18).
- No significant changes were observed in the response time of surface righting and negative geotaxis reflex (data not shown).
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Generation:
F2
Effect level:
5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 44.2 and 61.1 mg Al/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Generation:
F2
Effect level:
500 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 6.57 and 9.36 mg Al/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
>= 5 000 ppm
Based on:
test mat.
Remarks:
AAS; equivalent to 44.2 and 61.1 mg Al/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Table1: Absolute and relative organ weight of F1 and F2 male weanlings (% to the control)

AAS (ppm)   0     50     500     5000   
Organ weight  F1 males  F2 males  F1 males  F2 males  F1 males  F2 males  F1 males  F2 males
 number of animals  24  22  20  18  23  22  24  23
 body weigth (g)  100%  100%          85.97**  82.21*

 brain                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  113.59**  107.36*

 thymus                        

 absolute weigth (g)  100%  100%  NS  NS  NS  NS  76.79**  78.93**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  89.21*  86.09**

 liver                        

 absolute weight (g)  100%  100% NS NS   NS  NS  81.48**  89.56*
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  94.76*  NS

 kidneya                        

 absolute weigth (g)  100%  100%  NS  NS  NS  NS  90.74*  NS
rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  105.22*  107.96**

 spleen 

 
 absolute weight (g)  100%  100%  NS  NS  NS  NS  69.36**  74.86**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  80.76**  80.92**

 adrenala                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  90.91*  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

 testisa                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  90.02*  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

 epididymisa                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  84.01**  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

NS- no statistically significant differences were observed in the effect between Al treated and control animals

**- significantly different from the control, p<0.01; *- significantly different from the control, p<0.05;

a- value represent the total weights of the organs on both sides

Table2: Absolute and relative organ weights of F1 and F2 female weanlings (% to the control group)

AAS (ppm)   0   50     500     5000   
 organ weight  F1 females  F2 females  F1 females  F2 females  F1 females  F2 females  F1 females  F2 females
 number or animals  24  22  21  18  23  22  24  23
 body weight (g)  100%  100%          87.59**  90.50**

brain     

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  110 .88**  108.21**

 thymus      

 absolute weight (g)  100%  100%  NS  NS  NS  NS  82.72**  81.74**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

liver    

 absolute weight (g)  100%  100%  NS  NS  NS  NS  86.54**  84.73**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  93.49**

 kidneya   

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  107.96**  107.08

 spleen 

 absolute weight (g)  100%  100%  NS  NS  NS  NS  75.14**  77.75**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  85.57**  86.06**

 adrenala

 absolute weight (g)  100%  100%  NS  NS  89.01**  NS  87.45**  NS
 rel. weight (g/100g bw)  100%  100%  NS  NS    NS  NS  109.45*

 ovarya

 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  87.83**
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

 uterus

 absolute weight (g)  100%  100%  NS  NS  NS  NS  74.89**  75.35*
 rel. weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

NS- no statistically significant differences were observed in the effect between Al treated and control animals

**- significantly different from the control, p<0.01; *- significantly different from the control, p<0.05;

a- value represents the total weights of the organs on both sides

Conclusions:
The major findings reported by the authors were decreased body weight in preweaning animals, delayed maturation of the female offspring, and decreased organ weight in the offspring. However, because the effects could be related to decrease fluid consumption and limited nursing ability of dams, the utility of this study for risk assessment is limited.
Limited examination of the internal and external abnormalities, morphological variations and malformations precludes rigorous assessment of the teratogenic potential of the AAS test compound. As urinanalysis was not performed, possible adverse effects of prolonged AAS ingestion on kidney function cannot be assessed.
Executive summary:

Hirata-Koizumi et al. (2011b) investigated the potential reproductive toxicity of aluminium ammonium sulfate (CAS#: 7784-25-0 (anhydrous)) CAS#: 7784-26-1 (dodecahydrate)] in a GLP and OECD TG 416 -compliant 2 generation reproductive toxicity study. 

Aluminium ammonium sulphate (AAS) was dissolved in deionized water at 0, 50, 500 or 5000 mg/L. The Al concentration in the deionized water was < 5 µg/mL and the Al content of the diet was 22-29 mg/kg Groups of 24 male and 24 female Crl:CD (SD) rats (F0 generation) were given AAS in drinking water from 5 weeks of age for 10 weeks prior to mating, during mating and gestation, when the parental males were culled, and for the females through weaning. Litters were normalized to 8 pups on PND 4. At weaning, 24 males and 24 females were selected to serve as the F1 generation and they were given AAS in drinking water for 10 weeks prior to mating, during mating and gestation, and for the females through weaning, as for the F0 generation. Exposure of the F1 weanlings occurred at the same concentrations as those of their parents.

Spontaneous locomotor activity was assessed at 4 weeks of age in 10 male and 10 female randomly selected F1 pups per group. Rats were placed in transparent polycarbonate cages and observed using an infrared sensor. Observations were made at “10 minute periods and for a total of 60 minutes.” At 6 weeks of age a water-filled multiple T-maze was used to assess the spatial learning abilities of 10 male and 10 female F1 pups from each treatment group (selected randomly). Habituation of swimming ability was conducted by allowing the animals to swim three times in a straight channel the day before the trial. Testing was done in blocks of three trials on three consecutive days. Rats were restricted to 3 min in the water to prevent physical exhaustion, but whether any rats were excluded on the basis of their performance was not reported. The parameters recorded to assess performance included time from entry into the water, elapsed time to traverse the straight channel, reaching the “goal ramp” and the numbers of errors. 

Drinking water consumption was reduced at all concentrations compared to that of the concurrent controls. These reductions were clearly concentration-dependent and the reduction was significant at 500 and 5000 ppm in males and females of the F0 and F1 generations. These reductions were significant at 50 ppm in the F0 males and at some intervals during AAS exposure of the F0 and F1 females. 

A transient decrease in food consumption was observed in the 500 and 5000 ppm groups and in body weight in the 5000 ppm group. One F1 male in the 500 ppm group died, but that death was not considered treatment-related.

There were no significant effects of AAS consumption on the oestrus cycle. The authors reported no differences for copulation, fertility index, gestation index, precoital interval, gestation length, number of implantations, live pups delivered or delivery index, sex ratios of pups or viability during the preweaning period in females compared to the control. There were no significant differences between control and AAS-treated groups regarding the numbers of testis and cauda epididymal sperm, percentage of motile and progressively motile sperm, sperm swimming patterns and speed or the numbers of morphologically-abnormal sperm. Moreover, there were no significant differences in the numbers of primordial follicles in the F1 ovaries between animals given 5000 ppm AAS and those consuming deionized water. 

In the F1 and F2 pups, there were no treatment-related differences in numbers of offspring with congenital malformations, sex-ratio or viability on PND 0, 4 or 21. Reduced body weights were reported in the F1 male and female pups at 5000 ppm, but not in lower dose groups. The F1 male pups had a significantly lower body weight on PND 21, F1 female pups on PND 14 and 21, and F2 male and female pups on the PND 26. In female F1 pups, vaginal opening was delayed significantly among those whose mothers consumed 5000 ppm AAS (mean ± S.D: 32.3 ± 1.8 days vs. 30.2 ± 2.1 days in control), but their body weights were not significantly different from those of the concurrent control at the time of vaginal opening.

Absolute weights of testes and epididymis of the F1 and F2 male pups at 5000 ppm were lower than control. Absolute weights of the uterus were significantly lower in the F1 female pups, and absolute weights of the ovary and uterus were significantly lower in the F2 females. Histopathological examination revealed no treatment-related changes in the reproductive organs. Hirata-Koizumi et al. (2011b) considered these findings secondary to the decreased body weights and attributed the reductions in growth and development of the offspring “to the astringent taste of AAS which would decrease the palatability of drinking water in the AAS-treated groups”.

Spontaneous locomotor activity was no different among F1 males from dams given AAS in drinking water and those whose mothers consumed deionized water alone. There was some variation in activity among the F1 females.

The results presented by Hirata-Koizumi et al. (2011b) provide no evidence that prolonged consumption of AAS has an adverse impact on copulation, fertility and reproductive success in male and female Crl:CD(SD) rats consuming up to 517 mg AAS/kg-day. In discussing their data, Hirata-Koizumi et al. (2011b) concluded that “copulation, fertility or gestation indices were not affected up to the highest dose tested at which average Al intake from food and drinking water was estimated to be 36.3-61.1 mg Al/kg per day.” 

The authors identified a LOAEL of 5000 ppm AAS/L for both parental toxicity and reproductive toxicity (based on reduced preweaning body weight gain in F1 male (at PND 21) and female (PND 14, 21) pups, delay in the vaginal opening in F1 female pups, potentially attributed to inhibition of growth and decreased organ weights in F1 and F2 male and female offspring). The suggested LOAEL level corresponds to 36.3 mg Al/kg bw per day (Table 1, p.6). The reported NOAEL from the Hirata-Koizumi et al. (2011b) study is 500 ppm AAS/L which corresponds to 5.35 mg Al/kg bw per day (Table 1, p.6). 

Strengths of this study include the fact that it was conducted according to OECD TG 416,in according with GLP procedures, multiple dose levels were studied, both sexes were used, administered doses were analytically verified, a dose finding study was conducted and used doses were justified, stability of AAS in drinking solutions was detected and controlled.

Limitations of this study include a large gap (factor of 10) between the dose levels, lack of examinations/evaluation data on clinical signs of toxicity and absence of pH values for the AAS-containing drinking solution detract from the report. Few details were provided on the statistical methods used, blinding of observations, selection of the animals for the neurobehavioral tests, sperm parameters examination and the fact no measures of Al levels were conducted in the body fluids/organs. Possible irritant effects of AAS on the gastrointestinal tract mucosa of rats based on the results from the preliminary range-finding study could not be excluded. 

The Hirata-Koizumi et al. (2011b) study was assigned a Klimisch Score of 2.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008 - 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. (Spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
spacing of dose levels is greater than recommended on OECD 416, Al levels in blood and urine were not measured
Qualifier:
equivalent or similar to
Guideline:
other: Japanese guidelines for the "designation of food additives and for the revision of standards for the use of food additives".
Deviations:
not applicable
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: (P) 5 wks; (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing:
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 7 days

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

IN-LIFE DATES: From: To:
Route of administration:
oral: drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared at least every 6 days and kept under cool conditions until serving and drinking solutions were replaced at least once every 4 days
Details on mating procedure:
- M/F ratio per cage: 1:1 from the same treatment group
- Length of cohabitation: until successful copulation occured or the mating period of 2 weeks had elapsed
- Proof of pregnancy: during mating period vaginal smears were examined daily for the presence of sperm, presence of sperm in vaginal smears/or vaginal plug were considered as evidence of successful mating (day0 of gestation)
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: after day17 of gestation to day21 after delivery, wire-mesh floors of cages was replaced with stainless steel tray and individual dams or litters were reared using wood chipsas bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
during the study the, the concentrations of AS in drinking water were analyzed in the first and last preparations and once every 3 months, and verified using high performance liquid chromatography (quantitation limit 5 µg/mL)
Duration of treatment / exposure:
F0 males: 10 weeks prior to mating, during mating and up to parturition of the paired females
F0 females: 10 weeks prior to mating, during mating and during lactation until sacrifice after weaning of their pups (PND26)
F1: selected on PND 21-25 (designated as day0 of dosing) and were treated in same way as F0 males and females
Frequency of treatment:
animals were dosed up to the day prior to necropsy
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-25 days of age.
- Age at mating of the mated animals in the study: 13 weeks (F1), 15 weeks (F0)
Dose / conc.:
120 ppm (nominal)
Remarks:
For actual doses received see table1 under any other information on materials and methods including tables.
Dose / conc.:
600 ppm (nominal)
Remarks:
For actual doses received see table1 under any other information on materials and methods including tables.
Dose / conc.:
3 000 ppm (nominal)
Remarks:
For actual doses received see table1 under any other information on materials and methods including tables.
No. of animals per sex per dose:
24 animals per sex and group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a preliminary range-finding study
- Rationale for animal assignment: by stratified random sampling based on body weight
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, For dams body weight was recorded on gestational days 0, 7, 14 and 20 and days 0, 7, 14 and 21 of lactation (and additional day 4 of lactation for body weight)

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Time schedule for examinations: weekly. for dams, food consumption was recorded on gestational days 0, 7, 14 and 20 of gestation and days 0, 7, 14 and 20 of lactation (and additional day 4 of lactation for body weight)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice a week and on days 0, 7, 14 and 20 of gestation and days 0, 4, 7, 11, 14, 17, 19 and 21 of lactation
Oestrous cyclicity (parental animals):
Daily vaginal lavage samples were evaluated for each female for estrous cyclicity throughout the last 2 weeks of the premating period and during cohabitation until evidence of copulation was detected. Females with repeated 4-6 day estrous cycles were considered as having normal estrous cycles.
Sperm parameters (parental animals):
Sperm parameters were determined in all F0 and F1 adult males on day of sacrifice; right testis was used to count testicular homogenization-resistant spermatid heads; the right epididymal cauda was weighed and used for sperm analysis.
For sperm motility, the percentage of motile sperm and progressively motile sperm and the swimming speed and pattern were determined using a somputer-assissted cell motion analyzer (TOX IVOS). After recording sperm motion, the cauda epididymalfluid was diluted and the sperm were enumerated with a hematocytometer und a light microscope. Sperm count per gram of epididymal tissue was obtained by dividing the toal count by the gram weight of the caudal epididymis.
Sperm orphology was studied by stainign sperm with eosin and mount it on glass slides. 200 sperm in each sample were examined under light microscope and percentage of morphologically abnormal sperm was calculated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter ([4/sex/litter as nearly as possible, no adjustment was made for litters of fewer than eight pups); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
Number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities. clinical signs of toxicity (daily) and the body weight of live pups were measured on PND 0, 4, 7, 14 and 21.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

OTHER:
- developmental landmarks: Pinna unfolding in all F1 and F2 pups (PND1 - PND4); anogenital distance (AGD) measured on PND4 in all F1 and F2 pups; incisor eruption for one male and one female F1 and F2 pup selected from each liter were evaluated on PND8 and eye opening on PND12 until each pup fulfilled criteria
- neuromotor performances: surface righting reflex, negative geotaxis and midair righting reflex were assessed on PND5, 8 and 18 for one male and female F1 and F2 pup selected from litter
- neurobehavioral examinations:
locomotor activity - 10 male and 10 female F1 rats randomly selected from each group at 4 weeks of age (multi-channel activity monitoring system used)
T-maze test - water-filled multiple T-maze test was conducted in 10 male and 10 female F1 rats selected from each group at 6 weeks of age
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced (after a parturition of their paired females).
- Maternal animals: All surviving animals, after the last litter of each generation was weaned, on PND26.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
The number of uterine implantation sites was recorded for each dam. the testis and epididymis were prepared for microscopic examination and weighed. The brain, pituitary, thyroid, thymus, liver, kidneys, spleen, adrenals, testes, epididymis, seminal vesicles (with coagulating gland and their fluids), ventral prostate, uterus and ovaries in males and females were weighed before fixation, fixed and underwent macroscopic examination. The thyroid and seminal vesicles were weighed after fixation. In 10 F1 females, randomly selected from the control and highest dose group, the number of primordial follicles was counted in about 40 sections per ovary.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathologic evaluations were performed:
- in all animals of the control and the highest dose group
- in females with abnormal estrous cycle, abnormal delivery or total dead pups
- in males and females without evdence of copulation or insemination
- in all animals with grossly abnormal reproductive organs
- testes and epididymis were fixed in Bouin's solution and preserved in 70% ethanol; all other organs were fixed in 10% neutral bufferes formalin.

Testes, epididymis, seminal vesicles, ventral prostate, coagulating gland, ovaries, uterus and vagina were sectioned, stained with hematoxylin-eosin and examined under a light microscope. When treatment-related changes were found in the highest dose group, the same tissue from the next lower doese group then were examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 26 days of age.
- all pups found dead before weaning were necropsied immediately, following the adjustment of litter size on PND4, culled pups were sacrifized by carbon monoxide and subjected to gross external and internal examination

HISTOPATHOLOGY / ORGAN WEIGTHS
for one male and one female F1 and F2 weanlings selected from each dam:
- the brain, thymus, liver, kidneys, spleen, adrenals, testes, epididymides, ventral prostate, uterus and ovaries were removed and prepared for microscopic examination and weighed
- Since test substnace-related organ weigth changes were found in liver and spleen of highest dose group in F1 and F2 generations, these tissues were histopathologically examined for 10 male and 10 female F1 and F2 weanlings in the control and highest dose groups
- If treatment-related histopathological changes were observed in the highest dose group, the same tissue in the next lower dose group was examined as well.
Statistics:
Bartlett's test: was applied for homogeneity of distribution for parametric data (body weight, food and water consumption, length of estrous cycle and gestation, precoital interval, the number of implantations and pups born, delivery index, reflex response time, age atsexual maturation, behavioral test parameters, organ weight and sperm parameters);
For preweaning pups, body weight, AGD, viability, and age at the completion of developmental landmarks were similarly analyzed using the litter as the experimental unit.
One way analysis of variance was performed when the homogeneity of distribution was established.
If a significant difference was detected, Dunnett's test was conducted for comparison between control and individual treatment groups.
Data without homogeneity were analyzed using the Kruskal-Wallis rank sum test. If significant differences were found, the Mann Whitneys's U test was conducted fr comparison between the control and each dose group.
Fisher exact test was used to compare the incidence of parental animals with clinical signs, and autopsy and histopathological findings, the incidence of females with normal estrous cycle, incidence of weanlings with histopathological findings, copulation, fertility and gestation index, neonatal sex ration and completion rate of negative geotaxis between the AS and control group.
The Wilcoxon rank sum test was used to analyze the incidence of pups with clinical signs and necroscopy findings per litter, the completion rate of pinna unfolding in each litter, and the success rate of surface and mid-air righting reflex.
Student's T-test was used to compare the number of primordial follicles in the control and highest dose group because the homogeneity of variance was indicated by the F-test.
All of these statistical analysis were conducted using the 5% level of probability as the criterion for significance.
Reproductive indices:
- Copulation index (for males and females) (%): (no. of animals with successful copulation/no. of animals paired) x 100
- precoital interval (days)
- fertility index (for males and females) (%): (no. of males that impregnated a female or no. of pregnant/no. of animals with successful copulation) x 100
- Gestation index (%): (no. of females that delivered live pups/no. of pregnant females) x 100
- Gestation length (days)
- Delivery index (%): (no. of pups delivered/no. of implantations) x 100
- Estrous cycle in F0 and F1 females
Offspring viability indices:
For F1 and F2 offspring:
Maternal indices; no. of litters; no. of pups delivered; sex of all pups; sex ration of pups total (no. of male pups/total no. of pups)
Viability index calculated:
on PND 0 (%) = (no. of live pups on PND 0/no. of pups delivered) x 100
on PND 4 (%) = (no. of live pups on PND 4/ no. of live pups on PND 0) x 100
on PND 21 (%) = (no. of live pups on PND 21/no. of live pups on PND 4 after cull) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no significant difference seen between control and AS treated groups in incidence of clinical signs; 120 ppm: one F1 female died (non adverse); 600 ppm: one F0 female died (non adverse); 3000 ppm: one F1 femlae died (non adverse)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
food consumption was significantly decreased in 600 and 3000 ppm groups; body weight was decreased in 3000 ppm group
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: administered in drinking water
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant difference was seen between control and AS treated groups in the incidence of clinical signs of toxicity in either male or female P0 rats.
600 ppm: One P0 female died at 2 weeks of gestation. A subcutaneous mass was observed in the abdominal region of this female from the beginning of 5 weeks of dosing.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Drinking water consumption: P0 males and females - significantly lower than control throughout the study (120-3000 ppm)
Food consumption: P0 males - 600 ppm: significantly reduced during the first week of dosing; 3000 ppm: significantly decreased during weeks 1,8 and 13-14; P0 females - 600 ppm: significantly decreased during week 3 of lactation; 3000 ppm: significantly decreased at 1 week of dosing and during week 3 of lactation;
Body weight: P0 males and females - 3000 ppm: significantly decreased in the first 2 or 3 weeks of dosing

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
se table1 under "any other information on material and methody including tables"

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
P0 females - 120, 600 and 3000 ppm:
estrous cycle: no significant deviations in the estrous cycle of F0 females were observed during the premating period. However, a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
P0 males - 3000 ppm: absolute number of cauda epididymal sperm - reduced significantly (253.8 ±61.3 × 106/cauda versus 286.3 ±40.3 ×106/cauda in the control); however when expressed as the number per gram of tissues, there was no significant change. Number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm - no significant differences between control and AS-treated groups in P0 adults (Note: no details were provided on the results of these examinations).

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

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

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

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

OTHER FINDINGS (PARENTAL ANIMALS)
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
600 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 8.06 and 13.5 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Critical effects observed:
not specified
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant difference was seen between control and AS treated groups in the incidence of clinical signs of toxicity in either male or female P1 rats.
120 ppm: One P1 male died at 9 weeks of dosing. Soiling of periocular and perinasal fur and decreased locomotor activity were observed before death. Necropsy revealed accumulation of ascitic and pleural fluid and dark purple discoloration of liver and kidneys.
3000 ppm: One P1 male died at 12 weeks of dosing. No clinical signs of toxicity were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Drinking water consumption: P1 males - 120 ppm: significantly decreased during 3-6, 8, 10 weeks of dosing; 600, 3000 ppm: significantly decreased through dosing period; P1 females - 120 ppm: significantly decreased during 9-10 weeks of dosing; 600 ppm: significantly decreased during 10 week of dosing and 3 week of lactation; 3000 ppm: significantly decreased throughout the dosing period compared to controls
Food consumption: P1 males and females - 600 and 3000 ppm: significantly decreased in the 10 week of dosing (F1 males); significantly decreased in the 3 week of lactation (P1 females)
Body weight: 120 (P1 males), 600 (P1 males and P1 females) and 3000 ppm (F1 males and F1 females) - no significant differences in body weight compared to control; 120 ppm, P1 females - significantly increased body weights during 6-8 weeks of dosing

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
se table1 under "any other information on material and methody including tables"

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
P1 females - 120, 600 and 3000 ppm:
estrous cycle: no significant deviations in the estrous cycle of P1 females were observed during the premating period. However, a few control and AS-treated rats had persistent diestrus. The incidence of females with a normal estrous cycle also did not change significantly in either generation.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
P1 males - 3000 ppm: absolute number of cauda epididymal sperm - no change was found compared to the control animals. Number of testis sperm, the percentage of motile sperm and progressively motile sperm, the swimming speed and pattern, and the percentage of morphologically abnormal sperm - no significant differences between control and AS-treated groups in F1 adults (Note: no details were provided on the results of these examinations).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
P1 parental generation - copulation (males, females), fertility (males, females), gestation index, the precoital interval, gestation length, delivery index, the number of implantations, number of litters or pups delivered (see table 2)
- no significant differences were observed between the control and AS-treated groups in P1 generation.
Copulation was not observed:
- in the P1 males: in the control group (n=1), 120 ppm (n=2) 600 ppm (n=1) and 3000 ppm (n=3) and in the F1 females: in the 120 ppm group (n=1), 3000 ppm (n=1).
After successful copulation, no pregnancy was observed:
- in P1 females: in the control (n=2), 120 ppm (n=4), 600 ppm (n=2) and 3000 ppm (n=2).
No live pups delivered were found for pregnant rats from :
- P1 female in the 120 ppm group (n=1).
Comments: Overall, there were no treatment-related effects on reproduction parameters.

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

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

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

OTHER FINDINGS (PARENTAL ANIMALS)
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 38.5 and 55.6 mg Al/kg bw/day in P1 males and females, repsectively
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 38.5 and 55.6 mg/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
2 F1 showed malformations (non adverse), no malformed F2 pups were observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: F1 males and females - body weights were significantly lower on PND21; F2 females - body weights were significantly lower than controls on PND21; males - no significant differences
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: vaginal opening was significantly delayed (F1)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
3000 ppm: absolute and relative liver weights and absolute spleen weight, absolute weight of thymus, kidney, testes and epididymides decreased; absolute weight of uterus decreased at 600 ppm; relative brain weight decreased
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
F1 generation - 120, 600 and 3000 ppm:
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation.

CLINICAL SIGNS (OFFSPRING)
F1 generation - During the in-life check of delivered pups, one control F1 pup experienced trauma in the perianal region and tail and one F1 pup had hemimelia and oligodactyly in the 120 ppm group, but there was no significant difference between the control and AS-treated groups.

BODY WEIGHT (OFFSPRING)
F1 generation - 3000 ppm group, F1 males and F1 females
- body weights of male and female pups were significantly lower on PND 21 compared to the control.

SEXUAL MATURATION (OFFSPRING)
F1 males and females - 3000 ppm
- vaginal opening was significantly delayed (31.4±1.7 compared to 29.5±2.1 days in control). At 3000 ppm body weight at the time of vaginal opening was slightly higher than the control (119.0 ± 13.3 versus 109.6 ± 11.6 g) although not statistically significant.
- 120, 600 and 3000 ppm
- no significant differences between control and AS-treated groups were noted regarding age at preputial separation and no changes were found in body weights at the time of preputial completion.

ORGAN WEIGHTS (OFFSPRING) (see table3 and 4)
F1 generation - 3000 ppm, males and females
body weight - significantly lower at scheduled sacrifice compared to the control;
absolute and relative liver weights - significantly lower than the control;
absolute spleen weight - significantly decreased in both males and females and, a significant decrease in the relative weight was observed in males;
the absolute weight of the thymus - decreased in both sexes;
absolute weight of the kidney, testes and epididymides (males) - decreased compared to the control;
absolute weight of the uterus - decreased at 600 ppm compared to control;
relative brain weight - significantly increased in both sexes.

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

HISTOPATHOLOGY (OFFSPRING)
F1 males and females
- no dose-related histopathological changes in the liver or spleen of male and female F1 weanlings. (for details see table 3+4)

OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT:
F1 males and females; F2 males - 120, 600 and 3000 ppm
- the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening were not significantly different between the control and AS-treated groups.
F1 males and females - 120, 600 and 3000 ppm
- the AGD and AGD per cube root of the body weight ratio were not significantly different between control and AS-treated groups in male and female F1 and

NEUROMOTOR DEVELOPMENT
F1 males and females - 120, 600 and 3000 ppm
- no significant changes were observed in the development of reflexes (surface righting reflex on PND5, negative geotaxis reflex on PND8 and midair righting reflex on PND 18);
- no significant changes were observed in the response times of surface righting and negative geotaxis reflexes.

BEHAVIOR PERFORMANCE
F1 males and females - 120, 600 and 3000 ppm
Spontaneous locomotor activity was not significantly different between control and AS- treated groups at 10-min intervals and for 60 min.
Learning and memory performance in T-maze test
Pre-test swimming trials in the straight channel
- no differences between male and female rats in each group compared to the controls;
- no significant changes in the elapsed time to traverse the straight channel;
- in males, no significant changes in the elapsed time and number of errors on days 2–4;
- in females, the elapsed time and the number of errors was significantly lowered at 600 ppm on day 2, but there were no significant differences in the elapsed time or number of errors on days 3 and 4 between control and AS-treated groups (data not shown).
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Generation:
F1
Effect level:
3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Generation:
F1
Effect level:
600 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 8.06 and 13.5 mg Al/kg bw/day in P0 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 31.2 and 52.0 mg Al/kg bw/day in P0 males and females, respectively
Sex:
female
Basis for effect level:
sexual maturation
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
600 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 8.06 and 13.5 mg Al/kg bw/day in P0 males and females, respectively
Sex:
female
Basis for effect level:
sexual maturation
Critical effects observed:
not specified
VIABILITY (OFFSPRING)
120, 600 and 3000 ppm:
No significant changes were found in the viability index of pups at PND 0, 4, 21 in either generation.

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

BODY WEIGHT (OFFSPRING)
F2 generation - 3000 ppm, F2 females
- body weights were significantly lower than controls on PND 21.
3000 ppm, F2 males
- there were no significant differences in body weights between the control and AS-treated groups during the preweaning period.

SEXUAL MATURATION (OFFSPRING)
- 120, 600 and 3000 ppm
- no significant differences between control and AS-treated groups were noted regarding age at preputial separation and no changes were found in body weights at the time of preputial completion.

ORGAN WEIGHTS (OFFSPRING) (see table 3 and 4)
F2 generation - 3000 ppm, males
mean body weight at sacrifice - significantly lowered in both sexes;
absolute and relative weights of the thymus and spleen - significantly decreased in males;
absolute weight of the liver and epididymides - significantly decreased;
relative brain weight - significantly increased.
120 ppm, males
relative thymus weight - significantly decreased but no dose-response relationship.
3000 ppm, females
absolute and relative weights of the liver, the absolute weights of the spleen, ovary and uterus - significantly decreased;
relative brain weight - significantly increased.
600 ppm, females
the absolute brain weight - significantly decreased.

GROSS PATHOLOGY (OFFSPRING)
External and internal gross observations:
F1 males and females, F2 males and females
- no treatment-related alterations either in F1 and F2 weanlings or in pups found dead during the preweaning period (data not shown).

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

OTHER FINDINGS (OFFSPRING)
PHYSICAL DEVELOPMENT:
F2 males - 120, 600 and 3000 ppm
- the completion rate of pinna unfolding, and the age at completion of incisor eruption and eye opening were not significantly different between the control and AS-treated groups.
F2 males and females - 120, 600 and 3000 ppm
- the AGD and AGD per cube root of the body weight ratio were not significantly different between control and AS-treated groups in male and female F1 and F2 pups (data not shown).
F2 females - 120, 600 and 3000 ppm
-completion rates of pinna unfolding on PND 1, 3 or 4 and in other physical developmental landmarks were not significantly different between AS-treated groups and controls.
600 ppm - completion rates of pinna unfolding on PND 2 was significantly lower (17.0±35.4%, compared with 45.8±46.9 in controls), but no dose-response relation was observed.

NEUROMOTOR DEVELOPMENT
F2 males and females - 120, 600 and 3000 ppm
- surface righting reflex on PND 5 and negative geotaxis reflex on PND 8 were achieved in all male and female F2 pups in all groups;
- no significant changes were found in the response time (data not shown).
F2 females - 600 ppm
- the mid-air righting reflex on PND 18 was not achieved by 1 female in one of three trials; however, there was no significant difference in the mean success rate between the control and 600 ppm group (100±0.0% versus 98.4±7.3%).
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Generation:
F2
Effect level:
3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 38.5 and 55.6 mg Al/kg bw/day in P1 males and females, repsectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Generation:
F2
Effect level:
600 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 9.78 and 14.0 mg Al/kg bw/day in P1 males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
>= 3 000 ppm
Based on:
test mat.
Remarks:
Al2(SO4)3; equivalent to 38.5 and 55.6 mg Al/kg bw/day in P1 males and females, repsectively
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Table2: Reproductive performance of F0 and F1 parental animals

 AS (ppm)    0  120  600  3000
 F0 generation          
 No. of rats (male/female)    24/24  24/24  24/24  24/24
 Copulation index (%)  males  91.7  91.7  100  91.7
   females  95.8  100  100  100
 Precoital interval (days)    3.2 ± 1.1  3.2 ± 1.8  2.9 ± 1.3  2.8 ± 1.6
 Fertility index (%)  males  95.5  90.9  100  95.5
   females  95.7  91.7  100  95.8
 Gestation index (%)    100  95.5  95.7  95.7
 Gestation length (days)  22.4 ± 0.5  22.5 ± 0.6  22.1 ± 0.4  22.3 ± 0.5
 Delivery index (%)    94.3 ± 5.6  88.6 ± 21.0  90.7 ± 20.8  92.0 ± 20.5
 F1 generation          
 No. of rats (male/female)    24/24  23/24  24/24  24/24
 Copulation index (%)  males  95.8  91.3  95.8  87.5
   females  100  95.8  100  95.8
 Precoital interval (days)    3.3 ± 3.2  3.0 ± 2.0  2.7 ± 1.5  2.3 ± 1.1
 Fertility index (%)  males  91.3  81.0  91.3  95.2
   females  91.7  82.6  91.7  91.3
 Gestation index (%)    100  94.7  100  100
 Gestation length (days)    22.4 ± 0.5  22.3 ± 0.5  22.2 ± 0.4  22.2 ± 0.4
 Delivery index (%)    94.0 ± 9.9  87.5 ± 22.6  91.4 ± 10.7  94.6 ± 6.8

Table3: Absolute and relative organ weight of F1 and F2 male weanlings (% of control)

 As (ppm)  0     120     600     3000   
 Organ weight  F1 males  F2 males  F1 males  F2 males  F1 males F2 males  F1 males  F2 males
 number of animals  22  21  20  18  22  22  22  21
 body weight (g)  100%  100%  NS  NS  NS    87.44**  90.31**
 brain                        
 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
relative weight (g/100g bw)   100%  100%  NS  NS  NS  NS  113.22**  112.11**
 thymus                        
 absolute weight (g)  100%  100%  NS    NS  NS  81.33**  79.84**
 relative weight (g/100g bw)  100%  100%  NS  89.29*  NS  NS  NS  87.92**

 Livera                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  80.60**  87.78**
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  91.61**  NS
 Kidneya                        
 absolute weight (g)  100%  100%  NS  NS  NS  NS  89.62**  NS
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS
 spleen                         
 absolute weight (g)  100%  100%  NS  NS  NS  NS  76.40**  80 .43
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  86.93**  88.36**
 testisa                        
absolute weight (g)   100%  100%  NS  NS  NS  NS  90.44*  NS
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS
 Epididymisa                        
 absolute weight (g)  100%  100%  NS  NS  NS  NS  88.02**  93.62*
relative weight (g/100g bw)   100%  100%  NS  NS  NS  NS  NS  NS

NS- not statistically significant compared to untreated control

**- significantly different from control, p<0.05

*- significantly different from control, p< 0.01

Table4: Absolute and relative organ weight of F1 and F2 female weanlings (% of control)

 As (ppm)  0     120     600     3000   
 Organ weight  F1 females  F2 females  F1 females  F2 females  F1 females F2 females  F1 females  F2 females
 number of animals  22  22  20  18  22  21  21  21
 body weight (g)  100%  100%  NS    NS    89.91**  91.34**
 brain                        
 absolute weight (g)  100%  100%  NS  NS  NS  102.5*  NS  NS
relative weight (g/100g bw)   100%  100%  NS  NS  NS    110.20**  110.05**
 thymus                        
 absolute weight (g)  100%  100%  NS  NS  NS  NS  81.72**  NS
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

 Livera                        

 absolute weight (g)  100%  100%  NS  NS  NS  NS  84.80**  86.24**
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  94.26*  94.56**
 Kidneya                        
 absolute weight (g)  100%  100%  NS  NS  NS  NS  NS  NS
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS
 spleen                         
 absolute weight (g)  100%  100%  NS  NS  NS  NS  86.65**  84.11
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS
ovarya                       
absolute weight (g)   100%  100%  NS  NS  NS  NS  NS  84.52**
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

                       uterusa  

 absolute weight (g)  100%  100% NS NS   83.85*  NS  78.47**  81.49*
 relative weight (g/100g bw)  100%  100%  NS  NS  NS  NS  NS  NS

NS- not statistically significant compared to untreated control

**- significantly different from control, p<0.05

*- significantly different from control, p< 0.01

Conclusions:
Interpretation of the results is difficult due to the clear effect of AS treatment on fluid consumption. Addition of the test substance to drinking water at high concentrations led to reduced pH (3.57 to 4.2) and this appears to have reduced the palatability of the drinking water. At these AS levels, the F0 and F1 females also decreased their food consumption relative to the controls during week 3 of lactation. As a result, due to decreased drinking water consumption and decreased food consumption of F0 and F1 dams during the later stages of lactation, it is not possible to conclude with certainty whether the observations reported were associated with Al or represent secondary effects due to maternal dehydration and reduced nursing that may have influenced pup weight on PND 21. Because the effects reported could be related to decreased maternal fluid consumption, the utility of this study for risk assessment is limited.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
567 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the presence of a common metal ion, or ion complex including a hydrated metal ion, and following from this a similar chemical behaviour (refer to endpoint discussion for further details).
The available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no information available on the toxicity to reproduction of aluminium oxide.

Available data on the toxicity to reproduction/development of other aluminium compounds was taken into account by read-across following a structural analogue approach, since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+) (Krewski et al., 2007;). A detailed rationale and justification for the analogue read-across approach is provided in the technical dossier (see IUCLID section 13).

 

Overview of Epidemiological and Toxicological Studies

Studies of soluble aluminium compounds are relevant to this hazard assessment if it is assumed that, following oral exposure, the targeted aluminium compounds are solubilised in the gastrointestinal tract (GIT) in the presence of stomach and organic acids and that Al3+is an active moiety for systemic effects; it is recognized that the bioavailability of the sparingly soluble target compounds might be an order of magnitude less than these more soluble aluminium salts (Priest, 2010).

 

Human Studies

There are few human studies on the reproductive/developmental effects of ingested aluminium compounds. Several case studies have focused on children and pre-term infants receiving parenteral nutrition. A detailed discussion of these human case studies is presented in the comprehensive reviews by Krewski et al. (2007) and ATSDR (2008). 

Gilbert-Barness et al. (1998) reported the case of a girl who, at the age of 4 months, was diagnosed with severe mental retardation. A high Apgar score was allocated to the girl at birth and there was no recorded neonatal distress. Autopsy at age 9 revealed CNS cortical atrophy, small basal ganglia, and hypomyelination of the spinal cord, cerebral cortex, subcortex and cerebellar white matter. Later it was found that the mother had taken an average of 75 Maalox tablets (containing 200 mg of aluminium hydroxide per tablet) each day during pregnancy. It was suggested that the high levels of aluminium intake by the mother, during critical periods of the foetus’ brain development resulted in neurological damage to the infant (Krewski et al., 2007).

Bishop et al. (1997) reported that the Bayley index was significantly lower in the 39 pre-term infants who received more than 10 days of intravenous feeding of the standard feeding solution for pre-term infants than in the 41 pre-term infants who received more than 10 days of intravenous feeding of the Al-depleted standard solution. The standard and aluminium-depleted solutions delivered median daily aluminium intakes of 187 and 28 μg, respectively.

No statistically significant adverse pregnancy outcomes were observed in women accidentally exposed to high concentrations of aluminium sulphate in drinking water (concentrations were not specified in the paper) in northern Cornwall, England (Golding et al., 1991). The authors compared pregnancy outcomesin the affected area (n=68) after the incident with outcomes in a neighbouring unaffected area (n = 193). Except for a statistically significant increased prevalence of children showing talipes (4 cases vs. one control from the same area; p = 0.014), no exposure-related effects of aluminium were found with regard to perinatal deaths, low birth weight, preterm delivery, or severe congenital malformations. No follow-up studies have been conducted to investigate the possible long-term developmental effects in children born to mothers who were exposed to the high aluminium concentrations during pregnancy.

 

Animal Studies

Test guidelines for assessing reproductive endpoints include the Reproductive/Developmental Toxicity Screening study (OECD Test Guideline #421), the combined Repeated Dose Toxicity Study and Reproductive/Developmental Toxicity Screening study (OECD Test Guideline #422), the One–Generation Reproductive Toxicity Study (OECD Test Guideline #415), the extended One-Generation Reproductive Toxicity Study (OECD Test Guideline #443) and the Two–Generation Reproductive Toxicity Study (OECD Test Guideline #416). Of these, only the the extended One-Generation Reproductive Toxicity Study (OECD Test Guideline #443) provides adequate and complete information (ECHA, 2008, Chapter 7a) to meet the information requirements of REACH for an Annex X substance. A Two–Generation Reproductive Toxicity Study (OECD Test Guideline #416) that was initiated before 13 March 2015 are also considered appropriate to address the standard information requirement for this reproductive endpoint.

 

There are 2 two-generation studies available to support a hazard assessment of the reproductive effects of aluminium. However, interpretation of both studies for risk assessment is limited since effects reported may be due to limited water consumption seen in the study. Therefore, other OECD Test Guidelines can be used in combination to fulfill the information requirements. Currently, there are two GLP studies on reproductive/developmental toxicity of aluminium compounds available which are describe further down.

In the 2 OECD TG 416 and GLP compliant studies, aluminium sulfate Al2(SO4)3 (AS) and aluminium ammonium sulfate (AAS) (CAS#: 7784-25-0 (anhydrous)) CAS#: 7784-26-1 (dodecahydrate)] were administered by a relevant oral route with drinking water to Crl:CD(SD) rats at multiple dose levels (120, 600 and 3000 ppm and at 0, 50, 500 or 5000 ppm, respectively) before mating, during mating, gestation and lactation period in the two generation reproductive toxicity study (Hirata-Koizumi et al., 2011a+b). Twenty-four animals per sex and group (F0 and F1 generation) were given AS and AAS in pH 3.57 - 4.20 drinking water beginning at 5 weeks of age for 10 weeks until mating, during mating, throughout gestation and lactation. Litters were normalized on PND 4. In the F1 generation, 24 male and 24 female weanlings were identified as parents on PNDs 21 to 25, ensuring an equal distribution of body weights across groups. Drinking water provided to the F1 offspring contained the identical AS/AAS concentrations as those of their parents. These animals were then mated, and followed through gestation and lactation until sacrifice on PND 26. Each female was mated with a single male receiving the same AS/AAS drinking water concentration; if successful mating did not occur (as evidenced by sperm in a vaginal smear or presence of a vaginal plug) within the two week mating period, then the female was put in with another male from the same group who had mated successfully.

 

Observations assessed in the parental animals included clinical signs of toxicity, estrous cycle, copulation, fertility, gestation (including numbers of implantations) and delivery indices, the numbers of testis and cauda epididymal sperm, sperm swimming speed, percentage of motile sperm, percentages of motile sperm and percentages of morphologically abnormal sperm. Litter parameters recorded at parturition (post-natal day zero; PND0) included the number of live and dead offspring and the numbers and types of gross malformations. Developmental landmarks assessed in the F1 and F2 pups were: body weight (daily); sex ratios, pinna unfolding PND1 to PND4; anogenital distance on PND 4; incisor eruption (in one male and one female pup per dam) beginning on PND 8; eye opening beginning on PND 12; surface righting reflex (PND 5), negative geotaxis (PND 8); and mid-air righting reflex (PND 18) in one male and one female pup per litter. In the F1 pups selected as F1 parents, the males were observed for timing of preputial separation (starting on PND 35) and the females were observed for timing of vaginal opening (starting on PND 25). Neurobehavioral testing was conducted at two time points in randomly selected offspring (locomotor activity and T maze test).

The major findings in the aluminium sulfate study (Hirata-Koizumi et al., 2011a) include decreased drinking water consumption for both sexes in all AS groups, variable reductions in food consumption, reduced body weight in pre-weaning animals at 3000 ppm, delayed sexual maturation of the female F1 offspring at 3000 ppm, and decreased absolute liver, epididymides, thymus and spleen weight in the offspring at 3000 ppm. The authors proposed a LOAEL for aluminium sulfate for parental systemic toxicity and reproductive developmental toxicity of 31.2 mg Al/kg bw/day (3000 ppm) and NOAEL at 8.06 mg Al/kg bw/day (600 ppm). However, the authors state, correctly, that because “paired-comparison data are not available to assess the effects of decreased water intake in the absence of AS exposure” there is a possibility that the decreased absolute organ weights as well as delayed vaginal opening in the F1 females is likely secondary to the reduced body weight. The reduction in bodyweight is in turn likely to be related to the reduced food and water intake and a substance specific effect cannot be deduced from this study and the authors suggested their NOAEL was conservative.

 

The statistically significant delay in F1 female vaginal opening (29.5 ± 2.1 in controls and 31.4 ± 1.7 days in the highest dose group) was not accompanied by adverse changes in estrous cyclicity, anogenital distance or further reproductive performance. It is likely that the observed effects are secondary to the reduced body weight development. The authors concluded it is unlikely that “Al to have a clear impact on the hormonal event”. The AS levels added to drinking water by Hirata-Koizumi et al. (2011a) were 190, 946 and 4700 times greater than Al levels found naturally in drinking water (ca. 0.1 mg/L; WHO, 2003). 

The results presented on AAS (Hirata-Koizumi et al. 2011b) provide no evidence that prolonged consumption of AAS has an adverse impact on copulation, fertility and reproductive success in male and female Crl:CD(SD) rats consuming up to 517 mg AAS/kg-day. In discussing their data, Hirata-Koizumi et al. (2011b) concluded that “copulation, fertility or gestation indices were not affected up to the highest dose tested at which average Al intake from food and drinking water was estimated to be 36.3 - 61.1 mg Al/kg per day.” 

The authors identified a LOAEL of 5000 mg AAS/L for both parental toxicity and reproductive toxicity (based on reduced pre-weaning body weight gain in F1 male (at PND 21) and female (PND 14, 21) pups, delay in the vaginal opening in F1 female pups, potentially attributed to inhibition of growth and decreased organ weights in F1 and F2 male and female offspring). The suggested LOAEL level corresponds to 36.3 mg Al/kg bw per day. The reported NOAEL from the Hirata-Koizumi et al. (2011b) study is 500 mg AAS/L which corresponds to 5.35 mg Al/kg bw per day.

 

Interpretation of the results of both studies is difficult due to the clear effect of AS/AAS treatment on fluid consumption. Addition of AS to drinking water at high concentrations led to reduced pH (3.57 to 4.2) and this appears to have reduced the palatability of the drinking water. At these AS/AAS levels, the F0 and F1 females also decreased their food consumption relative to the controls. As a result, due to decreased drinking water consumption and decreased food consumption of F0 and F1 dams during the later stages of lactation, the observations reported represent secondary effects due to maternal dehydration and reduced nursing that may have influenced pup weight on PND 21. Because the effects reported could be related to decreased maternal fluid consumption, the utility of this study for risk assessment is limited.

 

A recent combined one-year developmental and chronic neurotoxicity study with Al-citrate (Alberta Research Council Inc, 2010) may be of interest for the evaluation of the neurotoxicity of Aluminium hydroxide, Aluminium metal and Aluminium oxide taking into consideration the tenfold lower bioavailability of Aluminium hydroxide, Aluminium metal and Aluminium oxide compared to Al-citrate and excluding effects that can likely be related to the salt rather than the cation. The study was conducted according to OECD TG 426 and GLP, and the exposure covered the period from gestation day 6, lactation and up to 1 year of age of the offspring. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions  via drinking water of  3225 mg/Al citrate/kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day). The highest dose was a saturated solution of Al-citrate. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L, equimolar in citrate to the high dose Al-citrate group) or plain water (control group). The Al citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Dams were sacrificed at PND 23. At PND 4  1 male and 1 female pup of each litter  were allocated to 4 testing groups: D23-sacrifice group for pre-weaning observations and D23 neuropathology, D64, D120 and D365 postweaning groups for post weaning observations and neuropathology at the respective days of sacrifice. Endpoints and observations in the dams included water consumption, body weight, morbidity and mortality and a Functional Observational Battery (FOB) (GD 3 and 10, PND 3 and 10). Pups were examined daily for morbidity and mortality. Additional neurobehavioral tests were performed at specified intervals and included, T-maze, Morris water maze, auditory startle, and motor activity. Female pups were monitored from PND26 for vaginal opening, male pups from day 35 for preputial separation. Clinical chemical and haematological analysis was performed for each group on the day of scheduled sacrifice. Al-concentrations were determined in blood, brain, liver, kidney, bone and spinal cord tissues by inductively coupled plasma mass spectrometric analysis. Further metals such as iron, manganese, copper and zinc were also determined. The pathological investigation includes rain weight and neuropathology. Statistical analyses were performed using the SAS software release 9.1. Data collected on dams and pups were analysed separately. All analysis on pups was performed separately for each sex. Statistical significance was declared from P ≤ 0.05.

Results: Dams: Eight high dose dams developed diarrhoea. In the Na-citrate group one dam stopped nursing and the pups were euthanized. No significant differences between mean body weights of dosed animals compared to controls were observed during gestation and lactation. During gestation and lactation low and mid dose group animals consumed considerably more fluid than controls and high dose group animals. This is not considered treatment related as there was no dose response. In all animals the target dose was exceeded during lactation due to the physiologically increased fluid consumption.

Pups: During the pre-weaning phase weights of mean body weights of male and females in the sodium citrate and high dose group were significantly lower than the untreated controls. This suggests a citrate rather than Al-related effect. No differences between treated and control animals were observed in the FOB. No other clearly treatment related effects were observed pre-weaning.

F1-postweaning: General toxicity

No significant differences in body weights throughout the study were observed between low and mid-dose animals sodium-citrate and untreated controls. High dose males had significant lower body weights than controls by PND 84. These animals also had clinical signs. At necropsy urinary tract lesions were observed in the animals of the high dose group, most pronounced in the males, hydronephrosis, uretal dilatation, obstruction and/or presence of calculi. All high dose males were sacrificed on study day 98. The effect is probably due to Al-citrate calculi precipitating in the urinary tract at this high dose level. This effect is related to the citrate salt and cannot be attributed to the Al-ion. Female high dose animals showed similar urinary tract lesions, but with a lower incidence and severity. Urinary tract lesions were also observed in single mid dose males, but also in a few sodium citrate and control animals. Fluid consumption during the study was increased in the sodium citrate and Al-citrate groups (in particular high and mid dose) compared to controls. This is probably due to the high osmolarity of the dosing solutions. However, the consumed dose levels decreased in all dose groups during the study. In the beginning the target dose was considerably exceeded, while versus the end of the study it was considerably below the target dose.  According to the authors the assigned dose levels still remain valid.

Developmental landmarks:

In sodium citrate controls and high dose males and females the number of days to reach preputial separation or vaginal opening was longer than in untreated control animals. This may be related to the lower body weights in these animals at the respective time-point. As the sodium citrate group showed similar retardation this effect cannot be allocated to the aluminium cation.

Neurobehavioral testing

No consistent treatment related effects that could be related to Al-ion exposure were observed in the FOB. No treatment related effects on autonomic or sensimotoric function were observed in the study. A weak association between Al exposure and reduced home cage activity, a very weak association with excitability, some association with neuromuscular performance were reported but according to the authors this may also be related to group differences in body weight, and an association with physiological function and is thus not considered clearly treatment related. No treatment related effect on general motor behavior was observed. No clearly treatment related effect on auditory startle response was observed. There was no evidence of any treatment related effect on learning and memory in the Morris Water Maze test and no clearly treatment related effects in the T-maze test. Hind limb grip strength and to a lesser extend foot splay were reported to be reduced compared to controls in high and mid dose male and female animals, more pronounced in younger than in older  rats. However, the observed effects can be related to the lower body weights of the individual animals undergoing this test. No details on the individual findings and historical control data are available. It can therefore not be concluded with certainty that the observed neuromuscular effects are primary effects of the treatment and attributable to Al3+. The NOAEL was reported based on this effect as 30 mgAl/kg bw in a conservative approach.

Haematology: No clinically significant differences in hematology were observed at the investigation on day 23. In day 64 and 120 females and day 64 males the high dose group showed slight reduction in hematocrit (males only), mean hemoglobin and mean corpuscular cell volume.No such changes were observed in the 364 day group.

Clinical chemistry: while a number of borderline statistically significant changes were observed, such as globuline levels, alkaline phosphatase and glucose in the high dose group little or no biological significance is associated with them. Elevated creatinine and urea levels in Day 64 males are consistent with the renal toxicity observed in these animals.

Organ weights: Brain weights did not differ among the groups, with two exceptions in the day 64 group males brain weights were significantly lower than controls. In the 120 day female high dose group brain weights were also significantly lower than controls. These findings were not reproduced at the other sacrifice times. Brains to body weight ratios were not significantly different and the lower brain weights can be attributed to the body weight.

Pathology: The main pathology findings were the renal lesions with precipitates in the urinary tract and secondary lesions such as hydronephrosis and uretal dilatation   in particular in the high dose group males and to a lesser extend females. Fluid colonic content was also observed in some high dose animals, in particular males. According to the authors the test item clearly precipitated in the urinary tract causing stone formation and blockage and resulted in fluid colonic content. No other macroscopic effects were observed in other organs.

Histopathology: No treatment related histopahological effects were observed in the nervous system at any time point.

Aluminium concentrations in different organs were dose related. Tissue concentrations were highest in blood, and then in decreasing order brainstem, femur, spinal cord, cerebellum, liver cerebral cortex.

A conservative NOAEL of 322.5 mg Al-citrate/kg bw  corresponding to 30 mg Al/kg bw was derived from this study. The most important effects were however related to a precipitation of the citrate in the kidneys and urinary tract and this effect is not related to the Al3+ ion.  The effects on grip strength and foor splay observed can also not be attributed unequivocally to Al-exposure as they may have been secondary to the general toxicity and body weight differences between treated and control animals undergoing this test. Neurobehavioral effects as reported by e.g. Thorne et al., 1986 could not be confirmed in this study.

 

In a GLP study, Beekhuijzen (2007) evaluated the effects of aluminium chloride (basic) (CAS# 1327-41-9) on early postnatal development in rats in a test study performed in accordance with OECD Test Guideline #422 (Combined Repeated Dose and Reproductive/Developmental Screening Test) [1]. Aluminium chloride (basic) was administered daily by gavage to male and female Wistar rats at doses of 0, 40, 200, 1000 mg/kg/day which contribute 0, 7.2, 36 and 180 mg Al/kg bw/day, respectively. Males were exposed to aluminium for 28 days, 2 weeks prior to mating, during mating, and up to termination; females were exposed for 37 to 53 days, 2 weeks prior to mating, during mating, during pregnancy and up to at least 3 days of lactation. Clinical signs of intoxication, mortality, body weights, food and water consumption, and reproduction process were recorded in both sexes. In addition, haematological and clinical biochemistry analyses were performed on both sexes at the end of study, together with macroscopic and microscopic examinations of the brain, thoracic and abdominal tissues and organs with special attention to the reproductive organs. Gross lesions were recorded for the cervix, clitoral gland, ovaries, uterus, and vagina in all female animals and the coagulation gland, epididymides, prepupital gland, prostate gland, seminal vesicles, and testes in all male animals. Body weights and the weights of the adrenal gland, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus were recorded for 5 animals from each group and sex. For each exposed group the following reproduction parameters were calculated: mating percentage (number of females mated x100/number of females paired); fertility index (number of pregnant females x100/number of females paired ); conception rate (number of pregnant females x100/number of females mated); gestation index (number of females bearing live pups x100/number of pregnant females); duration of gestation (number of days between confirmation of mating and the beginning of parturition); percentage of live males at first litter check (number of live male pups at first litter check x100/number of live pups at first litter check); percentage of live females at first litter check (number of live female pups at first litter check x100/number of live pups at first litter check); percentage of post-natal loss days 0 to 4 post-partum (number of dead pups on day 4 postpartum x100/number of live pups at first litter check) and viability index (number of live pups on day 4 postpartum x100/number of live pups at first litter check). The individual weights of all live pups on days 1 and 4 of lactation were measured and the sex of all pups determined by measuring the ano-genital distance. For offspring, clinical signs of intoxication and behavioural abnormalities were observed daily during at least 4 days of lactation.

No effects on developmental parameters in foetuses and offspring (growth, early development and survival) exposed to aluminium chloride (basic) at doses of 0, 40, 200 and 1000 mg/kg bw/day were reported. The NOAEL for reproductive toxicity (lack of effects on early development) proposed by the authors was 1000 mg/kg bw/day. A Klimisch Score of 2 was assigned to this study.

Results of other developmental toxicity studies in which prenatal, perinatal and/or post-weaning exposure of rats and mice to aluminium (as the hydroxide, chloride (basic), chloride, nitrate, and lactate) in the diet or drinking water were investigated are summarized below. 

 

Neurodevelopmental Deficits

Neurodevelopmental deficits have been reported in both mice and rats exposed via the oral route to aluminium at different life stages. The most commonly observed effects included decreased grip strength (Golub et al., 1992; 1995, Golub and Keen, 1999), reduced temperature sensitivity (Donald et al., 1989; Golub et al., 1992), reduced auditory startle responsiveness (Mishawa and Shigeta, 1993; Golub et al., 1994) and impaired negative geotaxis response (Bernuzzi et al., 1986; 1989; Muller et al., 1990; Golub et al., 1992). Decreased locomotor coordination, general motor activity level and impaired righting reflex have also been reported (Bernuzzi et al., 1986; Cherroret et al., 1992; Misawa and Shigeta, 1993). However, no treatment-related effects on locomotor activity and auditory startle response were reported in weanling male and female rats at the end of the lactation period following prenatal and postnatal (lactation) exposure to Al citrate (Alberta Research Council Inc, 2010). In the same study, no Al-citrate treatment-related effects were observed in the Functional Observational Battery tests performed on male and female rats at PND 5 and 11 (during the neonatal period) and on PND 22 (as juvenile pups).

Effects on developmental toxicity

Description of key information

Developmental toxicity, rat: NOAEL >= 768 mg/kg bw/day as aluminium hydroxide (equivalent to 266 mg Al/kg bw/day and 1004 mg Al oxide/kg bw/day)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
: lack of details on test substance
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Interfauna Iberica (Barcelona, Spain)
- Weight at study initiation: 225-240 g
- Diet: Food (commercial chow, Panlab , Barcelona, Spain), ad libitum
- Water: tap water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 °C
- Humidity (%): 45±5%
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h dark

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Details on the preparation of dosing solutions and on administration of the test substance are not provided.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data.
Details on mating procedure:
Females were housed with males (2:1) until copulation was detected.
Finding of sperm indicated copulation and the day of detection was designated day 0 of gestation.
Duration of treatment / exposure:
10 days (GD 6-15).
Frequency of treatment:
Daily (twice).
Duration of test:
20 days (GD 0-20).
Dose / conc.:
192 mg/kg bw/day (actual dose received)
Remarks:
Al(OH)3; equivalent to 66.5 mg Al/kg bw/day
Dose / conc.:
384 mg/kg bw/day (actual dose received)
Remarks:
Al(OH)3; equivalent to 133.0 mg Al/kg bw/day
Dose / conc.:
768 mg/kg bw/day (actual dose received)
Remarks:
Al(OH)3; equivalent to 266 mg Al/kg bw/day
No. of animals per sex per dose:
Twenty females for each dose group, 18-19 pregnant rats at termination.
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were randomly assigned to each experimental/control group.

Maternal-placental-fetal Al contents following gestational exposure have been examined. Al concentrations (µg/g wet weight) in maternal liver, brain, bone, placenta; and whole fetus (detection limit 0.05 µg/g).
Maternal examinations:
Clinical signs of toxicity, appearance, behavior changes were observed daily within 3 day intervals during pre-treatment, treatment and post-treatment period.
Hematological and serum biochemical analyses were performed on day 20 of gestation after euthanazy; blood was obtained by cardiac puncture.

Maternal body weight and food consumption:
Food consumption, maternal body weight and absolute/relative organ weight (kidney, liver, gravid uterine weight).

Post-mortem examination:
Animals sacrificed of GD 20.
Ovaries and uterine content:
Uterine content:
• Number of litters;
• corpora lutea;
• implantations/litter;
• preimplantation loss/litter;
• viable implants/litter;
• early resorption;
• late resorption;
• dead fetuses;
• postimplantation loss/litter.


Fetal examinations:
• Sex ratio;
• fetal body weight.
• Fetal examination for external and visceral abnormalities and skeletal malformations and variations performed.
Statistics:
• The unit of comparison was the pregnant female or the litter.
• Results of the quantitative continuous variables (e.g., maternal body weights, organ weights, fetal weights, etc.) were compared using analysis of variance (ANOVA) with significant F values further analyzed using Student’s t-test or Mann-Whitney U-test.
• Nonparametric data were statistically evaluated using the Kruskal-Wallis test when appropriate. The incidence of developmental abnormalities was not analyzed statistically because no differences were evident between treated and control groups.
Indices:
No data.
Historical control data:
No data.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No signs of toxicity, changes in body weight gain, absolute and relative internal organ weights (liver, kidney, gravid uterine) were observed in any group during the test (GD 0-20).

Group I
• increased number of early resorption (statistically non-significant).

Group II:
• >number of post-implantation loss (2.2 times);
• increased number of early resorption(not statistically significant).

Group III:
increased number of early resorption (statistically non-significant).

Statistically significant dose- and time-dependent decreased maternal food consumption during the treatment periods (6-15) was observed in Al treated animals compared to control group rats. However, food consumption recovered during the post-treatment period on GD 15-20.


No significant treatment-related differences were observed after exposure to Al hydroxide at any dose levels in:
• the number of corpora lutea;
• in percentage of pre-implantation loss ;
• in the number of total implants and viable; or nonviable implants per litter.
• > percentage of post-implantation loss – at 133 mg Al/kg bw/day (12.48± 11.56 vs 0.55 ± 0.90 in control group).

No dose-response observed for post-implantation losses. High variability was evident in all Al treated and control groups.

No Al related treatment effects were observed on maternal hematology and biochemical parameters at termination (GD 20) (data not shown).

No increased Al levels in maternal liver, bone, brain and placenta in Al treated animals compared to the control group was detected.

The maternal/placental Al concentrations were not statistically different between control and treated rats (high variability is evident in all groups).
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 266 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed at highest dose tested
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No differences were observed between control and Al treated groups in the incidence of individual malformations (external, visceral, or skeletal), or in the number of total malformations.

No significant changes in the incidence of any developmental variations were reported.

No increased Al levels in whole fetuses in any Al treated group (limit of detection – 0.05 μg/g.

Embryotoxic /teratogenic effects:
No Al treatment-related differences in number of live fetuses per litter, gender ratio, fetal body weight were detected.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 266 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed at highest dose
Abnormalities:
not specified
Developmental effects observed:
not specified
Executive summary:

The goal of study is to assess the developmental toxicity and embryotoxic/teratogenic potential of high doses of target compound - AI(OH)3orally administered to rats during the period of active organogenesis.

No significant general/maternal toxicity was observed in any Al treated groups that were orally exposed to Al hydroxide at doses 66.5, 133 and 266 mg Al/kg bw/day.

The results have contributed to the weight of evidence on the lack of pre-natal developmental toxicity of Al hydroxide administered orally to rats at high doses (66.6; 133 and 266 mg Al/kg bw/day). Toxicokinetic studies support the reported negative developmental outcomes.

Well designed and conducted study with many studied endpoints comparable to guideline OECD TG 414 with acceptable deviations.

However, particular test data/relevant information are absent (physiochemical properties, identification including CAS number, details on food and water quality (Al content in diet, water); details on test substance formulations (preparation, pH, stability and homogeneity of the preparation), details on reported negative teratogenic effects are not provided.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 004 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the presence of a common metal ion, or ion complex including a hydrated metal ion, and following from this a similar chemical behaviour (refer to endpoint discussion for further details).
The available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There is no information available on the toxicity to reproduction or development of aluminium oxide.

In terms of hazard assessment of toxic effects, available data on the toxicity to reproduction/development of other aluminium compounds was taken into account by read-across following a structural analogue approach, since the pathways leading to toxic outcomes are likely to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+) (Krewski et al., 2007;).

A few (five) developmental toxicity studies are available on aluminium hydroxide in mice and rats. In additon, a recent combined one-year developmental and chronic neurotoxicity study with Al-citrate is available (Poirier et al, 2011). A detailed rationale and justification for the analogue read-across approach is provided in the technical dossier (see IUCLID section 13).

Domingo et al. (1989) investigated the embryotoxic and teratogenic potential of Al (OH)3 administered orally to pregnant Swiss mice. Mated female mice (20 animals per group) were administered (oral, gavage) 0, 66.5, 133 or 266 mg Al(OH)3/kg bw/day (equivalent to 23, 46, and 92 mg Al/kg bw/day) from gestation day 6 through 15. Dams were sacrificed on gestation day 18. No sign of maternal toxicity was observed in any group based on changes in maternal weight gain, food consumption and gross signs of abnormalities at post-mortem examination. The number of total implantations, the foetal sex ratio, body weights and lengths of foetuses were not significantly affected at any of the administered doses of aluminium hydroxide. The number of early resorptions/litter was increased in all Al(OH)3 treated groups (3.0 - in the 23 mg Al/kg group, 2.4 - in the 46 mg Al/kg group, and 1.3 – in the 133 mg Al/kg group versus 0.4 in the control group) and the number of live foetuses decreased in all groups (11.1 in the control group, 9.4 in the 23 mg Al/kg group, 9.2 in the 46 mg Al/kg group and 9.8 in the 92 mg Al/kg group) (n = 18-20 litters per group). Observed effects were not considered as treatment related effects as there was no dose-response relationship observed. The Al-treated foetuses did not exhibit any marked differences in external malformations, internal soft-tissue anomalities or skeletal abnormalities compared to the controls. Suggested NOAEL is 266 mg Al/kg (lack of embryo/fetal toxicity or teratogenicity). The authors suggested that the lack of developmental toxicity of Al(OH)3 was likely due to lower gastrointestinal absorption of this compound compared with other forms of aluminium. A Klimisch Score of 2 was assigned to this study.

A similar study was conducted by Gomez et al. (1990) in rats. Aluminium hydroxide was administered by gavage (2 times, daily) to pregnant Sprague-Dawley rats at dose levels of 192 (n = 18 animals per group), 384 (n = 18 animals per group) and 768 (n = 10 animals per group) mg/kg (equivalent to 66.5, 133 and 266 mg Al/kg bw/day, respectively) from day 6 through 15 of gestation. The animals were killed on day 20 of gestation. No adverse effects were reported on animal appearance, behaviour, maternal body weight, or absolute and relative organ weight (uterine, kidney and liver). No differences were observed for haematological and biochemical parameters but detailed results for these outcomes were not provided in the publication. Although not statistically significant, the incidence of early resorptions was higher in all Al(OH)3-treated groups than in the control group (0.4 - in the 46 mg Al/kg group, 1.3 - in the 92 mg Al/kg group, and 0.6 – in the 266 mg Al/kg group versus 0.0 in the control group). Increased post-implantation loss (%) was observed compared to the control group (3.6 - in the 46 mg Al/kg group, 12.5 - in the 92 mg Al/kg group, and 5.0 – in the 266 mg Al/kg group versus 0.6 in the control group).Observed changes were not considered as treatment related effects because no relationship to dose was observed. Increased post-implantation loss (2.2 times compared to the control group) was observed only in the dose 92 mg Al/kg group. Statistically significant decrease in maternal food consumption was not associated with decreased maternal body weight and no dose-response relationship was found. No Al-treatment related effects were observed on critical gestational parameters such as number of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, or foetal body weight at any dose administered. During foetal examination, no external and visceral anomalities or skeletal malformation was detected. No significant differences in placental concentrations of aluminium were observed between the different groups. A Klimisch Score of 2 was assigned to this study. Suggested NOAEL is 266 mg Al/kg bw/day (lack ofembryo/fetal toxicity or teratogenicity).

The influence of citric acid on the embryonic and/or teratogenic effects of high doses of Al(OH)3 in rats was investigated by Gómez et al. (1991).Three groups of pregnant rats were administered daily doses (gavage) of Al(OH)3 (384 mg/kg bw/day, equal to 133 mg Al/kg bw/day , n = 18), aluminium citrate (1064 mg/kg bw/day, n = 15), or Al(OH)3 (384 mg/kg bw/day, equal to 133 mg Al/kg bw/day) concurrently with citric acid (62 mg/kg bw, n = 18) on gestational days 6 to 15. A control group received distilled water during the same period (n = 17). There were no treatment-related differences oncritical gestational parameters such as numbers of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, or foetal body weightin the group treated with Al(OH)3. No external and visceral abnormalities or skeletal malformation were detected on foetal examination. Maternal and foetal body weights were significantly reduced, the number of foetuses with delayed sternabrae and occipital ossification was significantly increased (p < 0.05), the number of foetuses with absence of xiphoides was increased in the group treated with Al(OH)3 and citric acid as compared to the control group. No significant differences in the number of malformations were detected between any of the groups (authors did not provide the quantitative data). A Klimisch Score of 2 was assigned to this study.

Colomina et al. (1992) evaluated the influence of lactate on developmental toxicity attributed to high doses of Al(OH)3 in mice. Oral (gavage) daily doses of Al(OH)3 (166 mg/kg bw, n = 11), aluminium lactate (627 mg/kg b, n = 10), or Al(OH)3 (166 mg/kg bw) with lactic acid (570 mg/ kg bw, n = 13) were administered to pregnant mice from gestational day 6 to 15.An additional group of mice received lactic acid alone (570 mg/kg bw).A control group (n = 13) received distilled water during the same period.No signs of maternal toxicity (no statistically significant changes in food consumption, maternal body and organ weight) were observed in the dams treated with Al(OH)3. No statistically significant treatment-related differences on critical gestational parameters such as number of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, or foetal body weight were observedin the Al(OH)3-treated group and no external abnormalities or skeletal malformation were detected on foetal examination. However, aluminium concentrations were significantly higher in the bones of dams, and aluminium was detected in the whole foetus of the Al(OH)3-treated animals. Concurrent administration of Al(OH)3 and lactic acid resulted in significant reductions in maternal weight compared to the control group. In the group given lactate only, aluminium was detected in whole foetuses; however, this was not statistically different from the mean level found in the control group. Aluminium lactate administration resulted in significant decreases in maternal body weight and food consumption, foetal body weight accompanied by increases in the incidence of cleft palate. Delayed ossification was also observed in the aluminium lactate-treated animals. Although not statistically significant, the incidence of skeletal variations was higher in the group concurrently administered Al(OH)3 and lactic acid than in the control group. No other signs of developmental toxicity were detected in the Al(OH)3 and lactic acid group. In the lactiv acid group no changes in maternal body weight were observed during the gestation period although food consumption was significantly decreased in early treatment (GD 6-9, P < 0.05; GD 6-15, P < 0.01) and post- treatment periods (GD15-18, P < 0.05).Additionally increased numbers of dead foetuses and litters with dead foetuses were seen but not significant.An increased number of foetuses with delayed ossification (10/4 vs 0/0 compared to control, P < 0.05) were also observed.A Klimisch Score of 2 was assigned to this study.

In a similar experiment, Colomina et al. (1994) assessed the effect of concurrent ingestion of high doses of Al(OH)3and ascorbic acid on maternal and developmental toxicity in mice.Three groups of pregnant mice were given daily doses (gavage, 2 times daily) of Al(OH)3 (300 mg/kg bw or 103.8 mg Al/kg), ascorbic acid (85 mg/kg bw), or Al(OH)3 concurrent with ascorbic acid (85 mg/kg bw) from gestational day 6 to day 15. A fourth group of animals received distilled water and served as the control group. The animals were killed on gestation day 18. Thenumber of litters, corpora lutea, number of total implantations, number of live foetuses, sex ratio, and foetal body weightdid not differ between the control and Al(OH)3-treated groups.No external and visceral abnormalities or skeletal malformations were detected on foetal examination. Placenta and kidney concentrations of aluminium were significantly higher in mice receiving Al(OH)3 and Al(OH)3 plus ascorbic acid than in controls.No information was provided on the number of dams and litters in the Al-treated and control groups. A Klimisch Score of 2 was assigned to this study.

In summary, available studies indicate that aluminium hydroxide did not produce neither maternal nor developmental toxicity when it was administered by gavage during the critical period of embryogenesis (GD 6-15) to mice at doses up to 92 mg Al/kg bw/day (Domingo et al., 1989) or to rats at doses up to 266 mg Al/kg bw/day (Gomez et al., 1990). The developmental toxicity of aluminium compounds following the oral route of exposure is highly dependent on the form of aluminium and the presence of organic chelators that influence bioavailability.

For all the studies with aluminium hydroxide, dose administration was by gavage, which would be expected to result in higher blood levels than dietary administration or administration via the drinking water, and very high dosages were used (ca. 200 – 2000x normal human exposure). Part of the reason for using such high dosages was the low solubility and bioavailability of aluminium hydroxide and the limited sensitivity of available analytical methods to determine small changes from endogenous levels of aluminium. However, the achieved dose of aluminium in maternal plasma was not measured in any of the studies reviewed. In the studies with aluminium administered in the diet or drinking water, dosages were generally identified in terms of the target dose, e.g. 1000 µg Al/g diet, without calculation of the actual dose administered based on the food or water consumption. Further, for the majority of the studies, there was no assessment of the background levels of aluminium in the food and water provided for the animals.

These factors generally lead to the conclusion that the dosages used in reproductive toxicity studies to date have been much greater than those that would be encountered in the human consumer or worker situation. In addition, the actual dose administered has usually been under-estimated because background aluminium levels in the diet and drinking water provided for the animals have not been taken into account. 

None of the studies on aluminium hydroxide showed any clear evidence of dose related developmental toxicity despite using daily dose levels up to 2000 fold higher than the normal aluminium levels of intake. Since bioavailability studies have shown that the absorption of aluminium oxide is less than that of aluminium hydroxide, it is unlikely that these would show any evidence of developmental toxicity at similar dose levels (Sullivan, 2010).

A recent combined one-year developmental and chronic neurotoxicity study with Al-citrate (Alberta Research Council Inc, 2010) may be of interest for the evaluation of the neurotoxicity of Aluminium hydroxide, taking into consideration the tenfold lower bioavailability of Al-hydroxide compared to Al-citrate and excluding effects that can likely be related to the salt rather than the cation. The study was conducted according to OECD TG 426 and GLP, and the exposure covered the period from gestation day 6, lactation and up to 1 year of age of the offspring. Pregnant Sprague-Dawley dams (n=20 per group) were administered aqueous solutions  via drinking water of  3225 mg/Al citrate/ kg bw/day (300 mg Al/kg bw/day); 1075 mg/Al citrate/kg bw/day (100 mg Al/kg bw/day); 322.5 mg/Al citrate/kg bw/day (30 mg Al/kg bw/day). The highest dose was a saturated solution of Al-citrate. Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L, equimolar in citrate to the high dose Al-citrate group) or plain water (control group). The Al citrate and Na-citrate were administered to dams ad libitum via drinking water from gestation day 6 until weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day (PND) 4. Weaned offspring were dosed at the same levels as their dams. Dams were sacrificed at PND 23. At PND 4  1 male and 1 female pup of each litter  were allocated to 4 testing groups: D23-sacrifice group for pre-weaning observations and D23 neuropathology, D64, D120 and D365 postweaning groups for post weaning observations and neuropathology at the respective days of sacrifice. Endpoints and observations in the dams included water consumption, body weight, morbidity and mortality and a Functional Observational Battery (FOB) (GD 3 and 10, PND 3 and 10). Pups were examined daily for morbidity and mortality. Additional neurobehavioral tests were performed at specified intervals and included, T-maze, Morris water maze, auditory startle, and motor activity. Female pups were monitored from PND26 for vaginal opening, male pups from day 35 for preputial separation. Clinical chemical and haematological analysis was performed for each group on the day of scheduled sacrifice. Al-concentrations were determined in blood, brain, liver, kidney, bone and spinal cord tissues by inductively coupled plasma mass spectrometric analysis. Further metals such as iron, manganese, copper and zinc were also determined. The pathological investigation includes rain weight and neuropathology. Statistical analyses were performed using the SAS software release 9.1. Data collected on dams and pups were analysed separately. All analysis on pups was performed separately for each sex. Statistical significance was declared from P ≤ 0.05.

Results: Dams: Eight high dose dams developed diarrhoea. In the Na-citrate group one dam stopped nursing and the pups were euthanized. No significant differences between mean body weights of dosed animals compared to controls were observed during gestation and lactation. During gestation and lactation low and mid dose group animals consumed considerably more fluid than controls and high dose group animals. This is not considered treatment related as there was no dose response. In all animals the target dose was exceeded during lactation due to the physiologically increased fluid consumption.

Pups: During the pre-weaning phase weights of mean body weights of male and females in the sodium citrate and high dose group were significantly lower than the untreated controls. This suggests a citrate rather than Al-related effect. No differences between treated and control animals were observed in the FOB. No other clearly treatment related effects were observed pre-weaning.

F1-postweaning: General toxicity

No significant differences in body weights throughout the study were observed between low and mid-dose animals sodium-citrate and untreated controls. High dose males had significant lower body weights than controls by PND 84. These animals also had clinical signs. At necropsy urinary tract lesions were observed in the animals of the high dose group, most pronounced in the males, hydronephrosis, uretal dilatation, obstruction and/or presence of calculi. All high dose males were sacrificed on study day 98. The effect is probably due to Al-citrate calculi precipitating in the urinary tract at this high dose level. This effect is related to the citrate salt and cannot be attributed to the Al-ion. Female high dose animals showed similar urinary tract lesions, but with a lower incidence and severity. Urinary tract lesions were also observed in single mid dose males, but also in a few sodium citrate and control animals. Fluid consumption during the study was increased in the sodium citrate and Al-citrate groups (in particular high and mid dose) compared to controls. This is probably due to the high osmolarity of the dosing solutions. However, the consumed dose levels decreased in all dose groups during the study. In the beginning the target dose was considerably exceeded, while versus the end of the study it was considerably below the target dose.  According to the authors the assigned dose levels still remain valid.

Developmental landmarks:

In sodium citrate controls and high dose males and females the number of days to reach preputial separation or vaginal opening was longer than in untreated control animals. This may be related to the lower body weights in these animals at the respective time-point. As the sodium citrate group showed similar retardation this effect cannot be allocated to the aluminium cation.

Neurobehavioral testing

No consistent treatment related effects that could be related to Al-ion exposure were observed in the FOB. No treatment related effects on autonomic or sensimotoric function were observed in the study. A weak association between Al exposure and reduced home cage activity, a very weak association with excitability, some association with neuromuscular performance were reported but according to the authors this may also be related to group differences in body weight, and an association with physiological function and is thus not considered clearly treatment related. No treatment related effect on general motor behavior was observed. No clearly treatment related effect on auditory startle response was observed. There was no evidence of any treatment related effect on learning and memory in the Morris Water Maze test and no clearly treatment related effects in the T-maze test. Hind limb grip strength and to a lesser extend foot splay were reported to be reduced compared to controls in high and mid dose male and female animals, more pronounced in younger than in older  rats. However, the observed effects can be related to the lower body weights of the individual animals undergoing this test. No details on the individual findings and historical control data are available. It can therefore not be concluded with certainty that the observed neuromuscular effects are primary effects of the treatment and attributable to Al3+. The NOAEL was reported based on this effect as 30 mgAl/kg bw in a conservative approach.

Haematology: No clinically significant differences in hematology were observed at the investigation on day 23. In day 64 and 120 females and day 64 males the high dose group showed slight reduction in hematocrit (males only), mean hemoglobin and mean corpuscular cell volume.No such changes were observed in the 364 day group.

Clinical chemistry: while a number of borderline statistically significant changes were observed, such as globuline levels, alkaline phosphatase and glucose in the high dose group little or no biological significance is associated with them. Elevated creatinine and urea levels in Day 64 males are consistent with the renal toxicity observed in these animals.

Organ weights: Brain weights did not differ among the groups, with two exceptions in the day 64 group males brain weights were significantly lower than controls. In the 120 day female high dose group brain weights were also significantly lower than controls. These findings were not reproduced at the other sacrifice times. Brains to body weight ratios were not significantly different and the lower brain weights can be attributed to the body weight.

Pathology: The main pathology findings were the renal lesions with precipitates in the urinary tract and secondary lesions such as hydronephrosis and uretal dilatation   in particular in the high dose group males and to a lesser extend females. Fluid colonic content was also observed in some high dose animals, in particular males. According to the authors the test item clearly precipitated in the urinary tract causing stone formation and blockage and resulted in fluid colonic content. No other macroscopic effects were observed in other organs.

Histopathology: No treatment related histopahological effects were observed in the nervous system at any time point.

Aluminium concentrations in different organs were dose related. Tissue concentrations were highest in blood, and then in decreasing order brainstem, femur, spinal cord, cerebellum, liver cerebral cortex.

A conservative NOAEL of  322 mg Al-citrate/kg bw  corresponding to 30 mg Al/kg bw was derived from this study (with a bioavailability correction this would correspond to ca. 300 mg Al from Al(OH)3).

The most important effects were however related to a precipitation of the citrate in the kidneys and urinary tract and this effect is not related to the Al3+ ion.  The effects on grip strength and foor splay observed can also not be attributed unequivocally to Al-exposure as they may have been secondary to the general toxicity and body weight differences between treated and control animals undergoing this test. Neurobehavioral effects as reported by e.g. Thorne et al., 1986 could not be confirmed in this study.

 

Inhalation Exposure

Overview of Epidemiological and Toxicological Studies

Aluminium metal, aluminium oxide and aluminium hydroxide

Human Studies

The effects of inhaled aluminium metal, aluminium oxide and aluminium hydroxide on reproductive/developmental outcomes have not been investigated directly in epidemiological studies.

Mur et al. (1998) reported a higher birth-rate among 692 French aluminium potroom workers who had always worked as potroom workers than among a control group of 588 male blue-collar workers who were employed in maintenance operations in the same 11 facilities. The control group had never worked in potrooms. Eligibility criteria to enroll in the study included: French nationality (to avoid cultural differences in sexual habits), marriage after entering the company (to ascertain the number of children born after the start of occupational exposure), and a length of employment of at least 1 year in the company, without any major change in the type of activity. The fertility data of the workers were obtained exclusively from the administrative files of the company. Based on the date of birth of the last child (dates of birth for all the children of each couple were not available), the annual birthrates of each couple after the marriage were calculated by dividing the total number of children of the couple by the number of years between the marriage date and the date of birth of the last child. No significant differences were observed between the ‘exposed’ and ‘control’ groups for the average year of marriage, the average age of the workers and of their spouses at the time of marriage, and the length of employment. Potroom workers were heavier smokers compared to the control group. The average number of live births in the potroom group was greater than that in the ‘control’ group; in addition, the average number of live births in both groups of aluminium industry workers was greater than the national average. After 30 years of marriage, the average numbers of live births were 2.63 (61.34) for the ‘control’ group, and 3.11 (61.74) for the ‘exposed’ group (P<0.05), the reference value for the entire French population being 2.28. The standardized birth ratio (SBR) in the control group was 1.04 (95% CI: 0.98 – 1.09) and 1.17 (95% CI: 1.12 – 1.23) in the potroom workers. The birthrate was higher in the exposed than in the unexposed group (birthrate ratio = 1.13; p < 0.001). The aim of this study was to evaluate the potential effects of occupational exposure to heat and static magnetic fields on male fertility. Exposure to aluminium compounds was not assessed. In addition, it was not possible to take into account a range of non-occupational and socio-economic factors that could influence fertility and birth-rate, for example income level, health status of male workers and their wives, and contraceptive practices. A Klimisch Score of 3 was assigned to this study.

 

Prasad et al. (2002) studied reproductive performance in 160 non-smoking aluminium foundry workers. These workers were engaged in melting aluminium ingots and alloying with magnesium and silicon followed by casting, rolling and coiling of aluminium wire, rods and conductors used for power transmission. The age range of the workers was 20 - 50 years and the duration of their employment in the factory ranged from 1 to 14 years. The exposed workers were compared with 150 male workers (control group) matched for age, smoking, drinking and socio-economic status with no occupational exposure to any known physical or chemical agents. Information on age, sex, duration of employment, health, medication, type of marriage (whether affinal or consanguineous), and reproductive history was collected by using a standard questionnaire. The reproductive parameters studied included the number of pregnancies in the workers’ wives, live births, stillbirths, abortions and the number of congenital defects, premature births, and neonatal deaths in their offspring. Air sampling was not undertaken in this study. There was no significant difference in fertility between the exposed and unexposed workers (99.33 versus 99.13, respectively, p < 0.05).There was a significant increase in the percentage of abortions (6.60% vs. 3.79%, compared to the control group, P < 0.05) and a decrease in the percentage of live births (89.28% vs. 93.94%, compared to the control group, P < 0.05) among the workers’ wives and more congenital defects in the offspring of the exposed workers than in the controls (1.03% vs. 0.03%, P < 0.05). Although there was an increase in the percentage of stillbirths and neonatal deaths in the offspring of the exposed, this increase was not statistically significant when compared with controls. No premature births were recorded in either the exposed or the control group. The authors mentioned that workers were exposed to polycyclic aromatic hydrocarbons, fluoride, fume of other metals, burnt gases, heat and static magnetic fields and high temperature. However, possible effects of these other hazardous compounds on male fertility were not assessed. Very limited details were provided on the study design and results. In addition, because of possible exposure to a range of confounding factors, the contribution of aluminium (from the foundry) to the reported adverse reproductive outcomes is unclear. A Klimisch Score of 3 was assigned to this study.

Hovatta et al. (1998) studied semen parameters (concentration, motility and morphology) and concentrations of aluminium, cadmium and lead in spermatozoa and seminal plasma from a group of workers in a refinery and a polyolefin factory (n =27, mean age 34 years, range 27 to 46 years)and a group of sperm bank donor candidates (n = 45, mean age 28 years, range 20 to 45 years).  The authors stated that the factories were located in a rural area of Finland with most of employees residing in the countryside while sperm bank donor candidates came from urban Helsinki. The concentration of aluminium in spermatozoa was lower in the group of employees than in the sperm donor candidates (0.93±0.69 (mean ± s.d. mg/kg) compared with 2.52 ± 4.14 mg/kg; p < 0.05). There was no significant difference between the groups with respect to aluminium content of seminal plasma. A weak but statistically significant inverse relation (Pearson r =-0.28; p<0.01) was observed between aluminium concentrations in the spermatozoa and sperm motility. A marginally significant inverse relationship was observed between sperm morphology and aluminium levels in the spermatozoa of men in the highest quartile of aluminium concentrations. Cadmium and lead levels did not show any statistically significant correlations with sperm parameters. The authors did not provide details of occupational exposures. Although the study provides some evidence for an association between aluminium levels in spermatozoa and sperm parameters, the small study size, the selected nature of the participants, and the lack of adequate characterization of possibly confounding occupational and environmental exposures limit its usefulness for hazard assessment. A Klimisch Score of 3 was assigned to this study.

 

Dawson et al. (1998) compared the levels of lead, cadmium and aluminium in relation to live sperm in semen samples from 64 healthy 21 to 35 year-old men. Spearman’s rank correlation between sperm viability and the semen plasma metal levels showed an inverse relation to aluminium (p < 0.01). The seminal plasma aluminium concentration was significantly higher in those with low sperm viability. Average concentrations were 1.01, 0.59 and 0.18 mg/L in the 18, 26 and 20 subjects with low, medium and high sperm viability, respectively. A Klimisch Score of 3 was assigned to this study.

Sakr et al. (2010) conducted a cross-sectional survey to examine reproductive outcomes in 710 active workers, both men and women, at a North America aluminium smelter. An anonymous questionnaire was developed to obtain information on the workers including age, level of education, occupational history and reproductive history (e.g., the pregnancies the workers had produced).  Participants were asked about the occupation of their partner during all pregnancies, the outcome of each pregnancy (pregnancy term, single live birth, multiple live birth, ectopic, abortion, spontaneous abortion, stillbirth, and molar), medical conditions experienced by mothers during the pregnancy (hypertension, diabetes, pre-eclampsia or eclampsia, thyroid disorder, systemic lupus, or other), age, smoking, and drinking habits. Normal live birth, miscarriage, live birth with congenital abnormally, and premature birth were selected for the analysis. Congenital anomalies were classified as major (an anomaly of surgical or cosmetic consequence) and minor (an anomaly with a little impact on individual well-being) by a nosologist (level of experience of a nosologist not provided) who was blinded to the employment status at the time of each pregnancy. All jobs at the aluminium smelter based on job titles were grouped into 3 categories: production, administration and laboratory. Random personal industrial hygiene samples for total dust, respirable dust, aluminium oxide, aluminium, asbestos, ammonia ,carbon monoxide, metallic and trivalent chromium, coal tar pitch volatiles as BSM, copper, cyanide (as CN), cyclohexane, fluorides (total), fluoride (particulate), fluoride gas (as HF), magnesium, manganese, metal and compounds, methanol, naphthalene, nickel, nickel compounds, RCF, crystalline silica, sulphur dioxide, and EMF were obtained through personal monitoring in the breathing zone of workers outside of any personal protective equipment.

To assess the occurrence of reported pregnancy outcomes among particular job categories, the authors identified reference groups in which all pregnancies had occurred during the pre-employment period (no details available on the selection and exclusion criteria of the reference groups). For each outcome, the proportion of pregnancies occurring during employment among the reference group was compared. The difference in proportions across employment groups was examined using the Chi square test. Logistical regression was used to account for the potential covariates. Data were stratified by gender. For the analysis of miscarriage, the data were stratified into pre-1999 and post-1999 due to increased awareness among workers in 1999 of the adverse pregnancy outcomes. The significance of the results was reported at P value less than 0.05.

The overall participation rate for the survey was 85% (621 of 730 workers); a higher proportion of women participated, 94% (106 of 113 workers) compared with 83% (515 of 617 workers) for men. All men and women who reported one or two pregnancies were included in the analysis (343 men and 76 women). The mean age at the time of the survey was 43.7 ± 6.3 years (mean ± SD) for men and 42.6 ± 7.3 for women. Most of men were involved in production-related jobs (80.5%) and 50% of the women held administrative positions. Most men had high school education (53.9%) whereas the majority of women had a college education (61.8%). Cigarette smoking and drinking were more prevalent before employment for both men and women.

The proportion of miscarriages reported by women and men was significantly lower in the pre-1999 than in the post-1999 period (76/759 or 10.01% vs. 37/160 or 23.13%; P<0.0001, respectively). Female workers had higher proportions of miscarriages than the spouses of male workers (34/184 or 18.48% vs. 79/735 or 10.75%, P=0.004, respectively). Working in the laboratory was significantly associated with the occurrence of congenital anomalies (OR, 7.89, CI 95% = 1.16 - 53.77). In women workers, the relationship between premature birth and most of the potential co-factors was not evaluated due to small number of reported cases (Table 3). For spouses of male workers, year of conception was also significantly associated with increased miscarriage (OR, 2.00, CI 95% = 1.05 to 3.80 for year of conception after 1999 compared to pre-1999 period). Working in a laboratory was associated with increased miscarriage for male workers but the difference in rate was not statistically significant (OR, 2.48, CI 95% = 0.74 to 8.31). For males, work in the production area was significantly associated with premature birth outcomes for their wives (OR, 2.85, CI 95% = 1.25 - 6.49). No statistically significant differences between rates of congenital anomaly in pregnancies fathered by male workers were reported. Overall, the results provide some evidence that both male and female workers of reproductive age and actively employed in the aluminium smelter experienced adverse reproductive outcomes during the period of employment (miscarriages, premature birth outcomes, and congenial abnormality). Prasad et al. (2002) reported increased congenial defects in offspring born to mothers/wives of the Al -exposed male workers employed at the aluminium foundry compared to the control group (P < 0.05); however, reported percentages were small (1.03% versus 0.03%, respectively). Since both female and male workers were exposed to other hazardous substances (burnt gases, SO2, coal-tar pitch volatiles, fluorides, etc.), in the workplace, the possibility that co-exposures to other toxicants resulted in the adverse effects cannot not be excluded. Study limitations include lack of data on the socio-economic status of the participants, previous exposure to hazardous substances, habits with regard to, and/or frequency of, use of contraceptive devices, and occurrence of genetic diseases in the families; also, the limited number of participants decreases the significance of reported findings. A Klimisch score 2 was assigned to this study.

Animal Studies

No adequate animal studies regarding the effect of the target aluminium compounds (aluminium metal, aluminium oxide and aluminium hydroxide) on developmental outcomes from exposure via inhalation were located. 

Other aluminium compounds

No histological changes were observed in reproductive organs and tissues (testes/ovaries, prostate/uterus, seminal vesicle) of Fischer 344 rats (male, female/10 animals per group) Hartley guinea pigs (male, females/10 animals per group) exposed by inhalation to 6.1 mg Al/m³ as aluminium chlorhydrate for 6 months, 6 hours per day, 5 days per week (Steinhagen et al., 1978). However, the authors did not examine reproduction function of the Al exposed animals.

Examination of reproductive function in male and female rats exposed by inhalation to 15.6±0.84 mg/m3of aluminium sulphate for 4 months did not reveal gonadotoxic or embryotoxic effects at the end of study (Grekhova et al., 1994).The authors provided only a very brief description of the study design and results which limits its reliability and usefulness for hazard assessment.

 

Oral exposure

It is likely that, once Al3+ has reached systemic circulation, its distribution is independent of the exposure route; therefore data obtained from studies of reproductive effects following oral exposure can be considered for assessment of this hazard following inhalation exposure. A few (five) developmental toxicity studies are available on aluminium hydroxide in mice and rats. For the exposure situations specified for the inhalation route, the most relevant studies are those in which either the target compounds have been administered (Domingo et al., 1989; Gomez et al., 1990; 1991; Colomina et al., 1992; 1994). A brief description of these studies and results is provided in the oral exposure section.

 

Weight of Evidence for Reproductive/Developmental Effects in Humans

Epidemiological studies of the effects of oral exposure to aluminium or its compounds on reproductive (developmenta) outcomes have not been conducted. The evidence from human studies is insufficient.

It is assumed that the aluminium ion, Al3+, is “the biologically active moiety” once the target substances are absorbed (following inhalation, ingestion or dermal contact) and “…likely to be similar or follow a similar pattern as a result of the presence of a common metal ion (or ion complex including a hydrated metal ion)” (Guidance on Grouping of Chemicals, OECD, 2007)[1].The cumulative weight of evidence based on the extensive database from animal studies examining various effects of soluble aluminium compounds on reproduction is modest. 

No reproductive toxicity studies are available for on the developmental toxicity of aluminium oxide or aluminium metal. Five studies on the developmental toxicity of aluminium hydroxide are available (Domingo et al., 1989; Gomez et al., 1990, 1991; Colomina et al., 1992, 1994). No clear evidence of dose-related developmental toxicity available based on these studies.

The weight of evidence for the association between exposure to aluminium metal, aluminium oxide and aluminium hydroxide and developmental toxicity in humans is limited.

Weight of Evidence for Reproductive Effects in Humans

Epidemiological studies of developmental toxicity associated with inhalation exposure to aluminium oxide, aluminium hydroxide and aluminium metal were not identified. The evidence from human studies is insufficient.

No animal studies were identified that investigated the effects of exposure to the the target aluminium compounds via inhalation on reproductive toxicity (developmental effects). As reproductive toxicity is a systemic effect, results from studies of effects from exposure via the oral route are relevant. Based on the reviewed animal studies, it is concluded that there is at presence no evidence of an association between inhalation exposure to the target aluminium compounds and developmental effects in both males and females.

Justification for classification or non-classification

According toRegulation (EC) No 1272/2008, classification as a reproductive toxicant is to be based on an assessment of the total weight of evidence. A weight of evidence approach is adopted in this document to identify of hazards to human health. Asubstantial number of studies using different animal models do not (unequivocally) support the association between oral exposure to Al and developmental effects (Krewski et al., 2007; WHO, 2007; EFSA, 2008; ATSDR, 2008; Health Canada, 2010).

Given that a critical factor influencing developmental toxicity is the concentration of the substance at the actual target site (ECHA, 2008, Chapter 7.12, p.148), “the human health hazard assessment shall consider the toxicokinetic profile (i.e. absorption, metabolism, distribution and elimination, ADME) of the substance” (Annex I, Section 1.0.2.). The occurrence and severity of reproductive effects of ingested aluminium compounds are a function of the bioavailability of the Al ion (Domingo, 1995; Golub & Domingo, 1996; Domingo et al., 2000; Krewski et al., 2007; ATSDR, 2008) and bioavailability is therefore relevant assessing the hazards of the target substances.Gastrointestinal absorption of the water soluble forms of aluminium compounds - aluminium nitrate nonanydate, aluminium chloride, and aluminium citrate - has been shown to be considerably higher than that of the sparingly soluble aluminium oxide, aluminium metal and aluminium hydroxide (Priest 2010).

Five reproductive toxicity studies with aluminium hydroxide were conducted with administration of high doses of aluminium hydroxide via the oral route of exposure. Collectively, these studies provide no clear evidenceof treatment-related developmental toxicity following oral exposure to aluminium hydroxide. A weight of evidence assessment based on the available reproductive toxicity studies with aluminium hydroxide does not support Classification and Labelling (EC No.1272/2008) requirements for developmental toxicity following oral exposure to aluminium hydroxide.

No studies on the reproductive toxicity of aluminium metal or aluminium oxide were located. However, the relatively similar aluminium bioavailability of all three targeted compounds (aluminium hydroxide, aluminium oxide and aluminium metal) following oral administration to laboratory animals (Piest, 2010) suggest that the availability of the aluminium ion for systemic effects will be similar for all compounds and aluminium metal and aluminium oxide also have a low potential to cause adverse developmental effects. 

 

Classification and Labelling for Adverse Health Effects on or via Lactation

In the current REGULATION (EC) No 1272/2008 on Classification and Labelling (page 109), effects on, or via, lactation are allocated to a separate single category; therein it is stated: “…….substances which are absorbed by women and have been shown to interfere with lactation, or which may be present (including metabolites) in breast milk in amounts sufficient to cause concern for the health of a breastfed child, shall be classified and labelled to indicate this property hazardous to breastfed babies”.

Classification can be assigned on the basis of:

(a) human evidence indicating a hazard to babies during the lactation period; and/or

(b) results of one or two generation studies in animals which provide clear evidence of adverse effects in the offspring due to transfer in the milk or adverse effect on the quality of the milk; and/or

(c) absorption, metabolism, distribution and excretion studies that indicate the likelihood that the substance is present in potentially toxic levels in breast milk (EC, No 1272/2008).

Based on available data, there is no conclusive evidence to suggest that aluminium absorbed bywomen can interfere with lactation (Yokel, 1984, 1985), and may be present in breast milk. However, currently, no data are available to confidently evaluate the toxicological significance and potential adverse health outcomes of aluminium levels found in breast milk (Krewski et al., 2007, p. 199).  

The magnitude of the contribution to the aluminium in breast milk from REACH-relevant exposures to aluminium metal, aluminium hydroxide or aluminium oxide is likely to be very small.

 

 

Based on the read-across from aluminium compounds for toxicity to reproduction or developmental toxicity, no classification is required according to DSD (67/548/EEC) or CLP (1272/2008/EC) classification criteria.