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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted with supporting substance, but according to OECD guideline 422 and GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
- Name of test substance as cited in study report: Aluminium chloride basic
- Physical state: Liquid (yellow)
- Stability in water: At least 48 hours
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Males approx. 9 weeks, females approx. 11 weeks
- Number of F0-animals: 40 males and 40 females
- Weight at study initiation: Males mean 266-269 g, SD 6.6-12.2; females mean 237-240, SD 7.0-10.7
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialitäten GmbH, Soest, Germany)
- Water: ad libitum, tap water
- Acclimatisation period: 4 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3 °C
- Humidity (%): 37-95 % (guideline requires 30-70%, but no effects on outcome expected)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark. Temporary fluctuations from the light/dark cycle (with a max. of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- Method of formulation: Formulations (w/w) were prepared within 4 hours prior to dosing and were homogenised 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.
- Frequency of treatment: Once daily for 7 days per week, approximately 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 necropsy.
- Dose volume: 5 ml/kg body weight. Acutal dose volumens were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulated samples were analysed using ICP-MS
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 37 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and durint at least 3 days of lactation.
Frequency of treatment:
1x daily, 7 days a week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3.6, 18, and 90 mg Aluminium/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40, 200, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
DOSE SELECTION RATIONALE:
Based on the results of a range finding study.

RANDOMISATION:
Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within +/-20% of the sex mean.

Examinations

Observations and examinations performed and frequency:
- Mortality/Viability: Checked at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating or housed individually. The time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales: grad 0 = absent, grade 1 = present (when maximum grade was 1); grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe (when maximum grade was 3 or 4). Cage debris of pregnant females was examined to detect abortion or premature birth. Signs of difficult or prolonged parturition were recorded.
- Functional observations: The following tests were performed in 5 males and 5 females randomly selected from each group: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system). During the motor activity test, males were caged individually and females were caged with their offspring. The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling). In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.
- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Following evidence of mating, body weights were recorded ong estation days 0, 4, 7, 11, 14, 17 and 20, on lactation days 1 and 4, and at death.
- Food consumption: Weekly, for males and females. Food consumption was not recorded during the breeding period. Following evidence of mating, food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20, and on lactation days 1 and 4.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investiation introduced as no effect was suspected.
- Reproduction processes: Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Clinical laboratory investigations: Blood samples were collected from 5 males and 5 females randomly selected from each group under iso-flurane anaestesia immediately prior to scheduled post mortem examination. Animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus. For determined parameters see section "Any other information on materials and methods incl. tables".
Sacrifice and pathology:
TERMINATION:
All animals were fasted overnight (with a maximum of 20 hours) prior to necropsy, but water was provided. All animals surviving to the end of the observation period and all moribund animals were anaesthetised using iso-flurane and subsequently exsanguinated. Males were killed on day 29 of study. Females were killed at day 4 post partum or shortly thereafter. Females showing no evidence of copulation were killed 24 -26 days after the last day of the mating period. In case a female was not pregnant, the uterus was stained using the Salewski technique in order to determine any very early post-implantation losses (= implantation site scars).

MACROSCOPIC EXAMINATION:
After sacrifice all parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paido to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. From all females, the number of implantation sites and corpora lutea were recorded.
Samples of the following tissues and organs were collected from 5 surviving animals per sex and group and fixed in 10% buffered formalin: Identification marks (not processed), adrenal glands, aorta, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), oesophagus, ovaries, pancreas, Peyer's patches (jejunum, ileum) if detectable, pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, thymus, thyroid including parathyroid (if detectable), trachea, urinary bladder uterus, vagina, all gross lesions. The following tissues/organs were not examined microscopically as no signs of toxicity of target organ involvement were indicated: eyes with optic nerve (if detectable) and Harderian gland, female mammary gland area, femur including joing, larynx, lacrimal gland (exorbital), nasopharynx, salivary glands (mandibular, sublingual), skeletal muscle, skin, tongue. The following tissues/organs were fixed in modified Davidson's solution: epididymides, eyes, testes.
From all remaining animals, the following tissues/organs were collected and fixed in 10% buffered formalin: Cervix, clitoral gland, coagulation gland, epididymides (fixed in modified Davidsons solution), ovaries, preputial gland, prostate gland, seminal vesicles, testes (fixed in modified Davidsons solution), uterus, vagina, all gross lesions.

ORGAN WEIGHTS:
The following organ weights (and terminal body weight) were recorded:
- From 5 surviving animals per sex and group: adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus
- From all remaining animals: Epididymides, testes
Statistics:
The following statistical methods were used to analyse the data:
- If the variables were assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Student's t-test (sokal, 1981) was applied for pup organ weights.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data was not assumed to follow a 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 as the lowest level of significance.
No statistical analysis was performed on histopathology findings. Additional methods of statistical analysis used and the results thereof are described in the report.
Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
See section "Details on results".
Mortality:
no mortality observed
Description (incidence):
See section "Details on results".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See section "Details on results".
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
See section "Details on results".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No mortality occured during the study period that was considered to be related to treatment. One control female was sacrificed in extremis due to parturition difficulties on day 22 of the post-coitum phase. This female delivered six dead pups. Necropsy revealed a total of 16 implantation sites in the uteri, and one dead foetus in the left uterus horn. No cause of moribundity could be established microscopically. All other rats survived the scheduled duration of the study.
There were no clinical signs of toxicity noted over the observation period.
Incidental findings that were noted consisted of salivation and alopecia of various body parts. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted in control males, males at 40 mg/kg bw/day and in both sexes at 200 mg/kg bw/day.

FUNCTIONAL OBSERVATIONS:
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
A slightly lower mean body weight was recorded for females at 1000 mg/kg bw/day on day 8 of the pre-mating phase, with slight weight loss for three high dose females. mean body weights of high dose females remained slightly lower during the mating phase and during the first week of the post coitum phase, but recovered towards control levels as treatment progressed. A level of statistical significance was achieved in these instances.
Slight weight loss was also noted for one high dose male during the pre-mating phase, but the mean body weight of this group remained similar to control levels throughout the study period. Therefore, no toxicological significance was ascribed to this incidental occurence.
Body weights and body weight gain of other treated animals remained in the same range as controls over the study period.
Mean food consumption of females at 1000 mg/kg bw/day was slightly lower than controls before or after allowance for body weight during the first week of the pre-mating phase.
Food consumption of other treated groups was similar between treated and control animals, before or after allowance for body weight.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
Reproduction parameters were unaffected by treatment up to 1000 mg/kg bw/day. Mating performance, duration of gestation, fertility parameter, number of corpra lutea, number of implantation sites, and number of dead and living pups at first litter check were similar for the control and treated groups. See also tables on reproduction data in section "Any other information on results incl. tables".
Breeding parameters were unaffected by treatment up to 1000 mg/kg bw/day. Postnatal loss between days 0 and 4 post partum were similar for the control and treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS):
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. The slightly lower brain weight of females at 1000 mg/kg bw/day was considered to have achieved statistical significance by chance, as the change was of minor nature and within the range expected for rats of this age and strain. Also, when corrected for terminal body weight, no statistical significance was achieved. The statistically significant lower kidney weights of females at 200 mg/kg bw/day occurred in the absence of a dose-related response. These minor changes were therefore considered not to be a sign of toxicity.

GROSS PATHOLOGY (PARENTAL ANIMALS):
Red foci were noted on the glandular mucosa of the stomach of 5/10 males at 1000 mg/kg bw/day, along with thickening of the glandular mucosa or limiting ridge in two of these cases.
In the control female sacrificed in extremis, dark red contents in the caecum, and enlargement of the spleen and liver were noted. Incidental findings among other control and/or treated animals are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance. These findings included a reduced size of the left thigh muslce, enlarged mesenteric or mandibular lymph nodes, red discolouration of the mesenteric lymph nodes, a tan focus on the right lateral lobe of the liver, watery-clear cysts on the kidneys, pelvic dilation of the kidneys, alopecia, red foci on the thymus and fluid in the uterus.
No macroscopic abnormalities were noted among males at 200 mg/kg bw/day and females at 1000 mg/kg bw/day.

MICROSCOPIC EXAMINATION (PARENTAL ANIMALS):
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 of both sexes at 1000 mg/kg bw/day.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
The incidence of suspected infertility among the animals did not indicate a relationship to treatment. In the females suspected of infertility, the following histopathological findings were noted: uterine horn decidual thickening indicating previous pregnany (in one control and one 40 mg/kg animal), uterine dilatation and trilaminar vaginal epithelium indicative of an active oestrus cycle (in one 40 mg/kg animal), moderate focal inflammatory haemorrhagic ulceration suggestive of a separated placentation (in one control animal), no indication of past or present reproductive activity (in one 200 mg/kg animal).
No abnormalities were seen in the reproductive organs of males suspected of infertility.
The control female sacrificed in extremis did not show any lesions that could be associated with a cause of morbundity.
All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occured at similar incidences and severity in both control and treated rats.

HAEMATOLOGY (PARENTAL ANIMALS):
The following minor, but statistically significant changes distinguished animals at 1000 mg/kg bw/day from control animals:
- Lower mean corpuscular haemoglobin concentration (MCHC) in both sexes
- Higher platelet counts in males
Individual increases of haematology parameters at 1000 mg/kg bw/day were not related to treatment-related morphological alterations. These changes included increased red cell distribution width (RDW) and reticulocyte counts of 2 animals, and increased relative eosinophilic counts of 2 other animals. Since a group response for these parameters was absent and the changes were of amild nature, no toxicological significance was ascribed to these alterations.
No dose-related response was apparent for the statistically lower haemoglobin levels of males at 40 mg/kg bw/day and higher, and for the changes in white blood cell counts of males and females at 40 mg/kg bw/day. Means remained within the range expected for rats of this age and strain. These changes were therefore considered to have arisen by chance and not to represent a change of toxicological significance.

CLINICAL BIOCHEMISTRY (PARENTAL ANIMALS):
The following statistically significant changes distinguished treated males from control animals:
- Lower alkaline phosphatase activity (ALP) levels at 1000 mg/kg bw/day
- Lower total protein levels at 1000 mg/kg bw/day
- Lower albumin levels at 1000 mg/kg bw/day
- Higher potassium levels at 200 and 1000 mg/kg bw/day
- Higher inorganic phosphate levels at 1000 mg/kg bw/day
Clinical biochemistry parameters of females were similar to control levels.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Local effects (stomach) at 1000 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
18 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
Al3+
Sex:
male
Basis for effect level:
other: Local effects (stomach) at 90 mg Al/kg bw/day
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Local and systemic (HISTOPATHOLOGY EFFECTS at 1000 mg/kg/day)
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
Al3+
Sex:
female
Basis for effect level:
other: Local and systemic (HISTOPATHOLOGY EFFECTS at 90 mg Al/kg/day)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with aluminium chloride basic by oral gavage in male and female Wistar rats at dose levels of 40, 200 and 1000 mg/kg bw/day revealed parental toxicity at 1000 mg/kg bw/day, comprising local stomach effects. Based on the findings on the stomach observed macroscopically, the parental NOAEL for local effects was established at 200 mg/kg bw/day. A parental NOAEL for systemic toxicity of 1000 mg/kg bw/day was established.
Executive summary:

Aluminium chloride basic was administered by daily oral gavage to male and female Wistar rats at dose levels of 40, 200 or 1000 mg/kg bw/day, corresponding to 3.6, 18 and 90 mg/kg bw/day of elemental aluminium. Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termintation. Females were exposed for 37 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 3 days of lactation.

Histopathological assessment revealed a mild to moderate subacute inflammation of the glandular stomach mucosa and minimal to moderate superficial mucosal eosinophilic spheroids in both sexes at 1000 mg/kg bw/day. In males, these findings supported the macroscopic findings of the glandular mucosa/limiting ridge at this dose level. The mucosal eosinophilic spheroids are apparently intracellular degenerative products of the superficial mucosa and possibly associated with the inflammation below the base of the mucosa. These findings are indicatie of local irritating properties of the test substance.

The slightly lower body weight and food intake of females at 1000 mg/kg bw/day recovered to control levels as treatment progressed. No toxicological significance was therefore ascribed to these changes.

Changes in clinical pathology parameters at 1000 mg/kg bw/day were of slight nature and generally within the range expected for rats of this age and strain. Also, any morphological correlates were absent. Therefore, these changes were considered not indicative of organ dysfunction and to be of no toxicologcal significance.

There were no further changes for mortality, clinical signs, functional observations and organ weights that were considered to be an effect of treatment.

The following NOAELs were established:

- NOAEL (parental animals) for systemic toxicity: 1000 mg/kg bw/day

- NOAEL (parental animals) for local effects on the stomach: 200 mg/kg bw/day