Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
7 September 2006-3 November 2006 (Report, 2007, p.11).
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions Al3+is the predominant aluminium form at low pH (less than 5.5). As pH increases above 5.5, aluminium-hydroxide complexes formed by hydrolysis become increasingly important and dominate aqueous aluminium speciation . Al3+, depending on the composition of the medium and pH, may form Al(OH)3 or Al2O3 or AlCl or AlNO3 or AlSO4. According to the solubility studies, the major elements found at the maximum solubility are 1054 mg/l Al and 856 mg/l Ca, for 100 g of the tested substance, ie approximatively 1%. By oral route, Aluminium chloride could be formed in the stomach, thus a read-across was made with this salt.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
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. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Aluminium chloride basic.
IUPAC Name:
Aluminium chloride basic.
Constituent 2
Reference substance name:
Aluminum chloride, basic
EC Number:
215-477-2
EC Name:
Aluminum chloride, basic
Cas Number:
1327-41-9
IUPAC Name:
aluminum trichloride
Details on test material:
- Name of test material (as cited in study report): Al(OH)13Cl17(in aqueous solution)
- Molecular weight (if other than submission substance): 109.36
- Composition of test material, percentage of components: Al (Al2O3) - 17.0%; Aluminium - 9.0%; Chloride - 19.9%; Arsenic < 0.1 mg/kg; Lead - 0.17 mg/kg; cadmium < 0.01 mg/kg; Chromium -0.29 mg/kg; Nickel - 0.36 mg/kg; Mercury < 0.01 mg/kg; Selenium < 0.1 mg/kg; Antimony < 0.1 mg/kg bw
- Physical state: yellow liquid
- Lot/batch No.: 5-510-37
- Expiration date of the lot/batch: 29 January 2006
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark
- Other:
Specific gravity: 1.36 g/cm³
pH: 1 at concentration of 100 M-%
Stability in vehicle/water: at least 48 hours
Solubility in water: 1-100%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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.


Administration / exposure

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.
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
other: 0 mg/kg bw, 40 mg/kg bw, 200 mg/kg bw, 1000 mg/kg bw of Al chloride basic; Assuming the substance is 18% Al by mass: 0, 7.2, 36, 180 mg/kg bw
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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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 (Appendix 1, p.2; Appendix 6, p. 11).
Increased salivation and excretion were noted in both females and males exposed to 1000 mg/kg of Al chloride basic (Appendix 1, p.2; Appendix 6, p. 11).


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 (Appendix 1, p.14).

* - 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” (Report, 2007, p. 26).

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 (Appendix 1, 2007, p.25)

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%)

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Remarks:
(rat, irritation of stomach mucosa)
Effect level:
1 000 mg/kg bw (total dose)
Sex:
male/female
Basis for effect level:
other: I. Irritation Effect on GI (local)
Dose descriptor:
NOAEL
Remarks:
(rat, irritation of stomach mucosa)
Effect level:
200 mg/kg bw (total dose)
Sex:
male/female
Basis for effect level:
other: I. Irritation Effect on GI (local)
Dose descriptor:
NOAEL
Remarks:
rat, lack of effects on reproductive, breeding and mating activity)
Effect level:
1 000 mg/kg bw (total dose)
Sex:
male
Basis for effect level:
other: II. Overall Reproductive toxicity
Dose descriptor:
NOAEL
Remarks:
rat, lack of effects on reproductive, breeding and mating activity)
Effect level:
1 000 mg/kg bw (total dose)
Sex:
female
Basis for effect level:
other: II. Overall Reproductive toxicity

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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 (Appendix 1, p.27).

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 (Appendix 1, p.28).

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).

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
II. Overall Reproductive toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
other:
Sex:
male/female
Basis for effect level:
other: Effect on early postnatal development Based on the reported results, suggested NOAEL (rat /males, females/ lack of early post-natal developmental toxicity) for reproductive toxicity of 1000 mg /kg bw of Al chloride basic.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL for reproduction, breeding and early post-natal developmental toxicity was suggested at a level of 1000 mg/kg bw.
Executive summary:

This GLP study was performed in accordance with OECD Test Guideline (TG) 422. Male and female Wistar rats were exposed to Al chloride basic at dose levels of 40, 200, and 1000 mg/kg body weight.

No mortality or clinical signs of intoxication were observed due to treatment with Al chloride basic.

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.

Conclusion : 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.