Aluminium sodium tetrahydroxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

07 Sep - 03 Nov 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Cross-reference

Reason / purpose for cross-reference:

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

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Temporary deviations from maximum level of relative humidity. Acclimatisation period 4 days instead of 5 days. Age at study initiation 9 (males) and 11 (females) weeks instead of ca. 10. The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Remarks:

NOTOX B.V., 's-Hertogenbosch, The Netherlands

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aluminum chloride, basic

EC Number:

215-477-2

EC Name:

Aluminum chloride, basic

Cas Number:

1327-41-9

Molecular formula:

General formula: Al(OH)x(Cl)(3-x), with x ranging from 0.1 to 2.3

IUPAC Name:

aluminum trichloride

Details on test material:

- Name of test material (as cited in study report): Aluminium chloride basic
- Molecular formula (if other than submission substance): Al(OH)1.3Cl1.7 (in aq. solution)
- Molecular weight (if other than submission substance): 109.36
- Physical state: yellow liquid
- Analytical purity: no data
- Composition of test material, percentage of components: Aluminium (Al2O3) 17%, Aluminium (Al) 9%, Chloride (Cl) 19.9%, Arsen (As) <0.1 mg/kg, Lead (Pb) 0.17 mg/kg, Cadmium (Cd) <0.1 mg/kg, Chromium (Cr) 0.29 mg/kg, Nickel (ni) 0.36 mg/kg, Mercury (Hg) <0.01 mg/kg, Selenium (Se) <0.1 mg/kg, Antimony (Sb) <0.1 mg/kg
- Lot/batch No.: 5-510-37
- Expiration date of the lot/batch: 29 Jan 2006
- Stability under test conditions: at least 48 h stable in vehicle (water)
- 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-%
Solubility in water: 1-100%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males ca. 9 wk, females ca. 11 wk
- Weight at study initiation: males ca. 267 g, females ca. 238 g
- Housing:
Pre-mating: 5 animals/sex/cage in Macrolon plastic cages (MIV type, 18 cm height).
Mating: Females and males caged together on a one-to-one basis in Macrolon plastic cages (MIII type, 18 cm height).
Post-mating: 5 males/cage in Macrolon plastic cages (MIV type, 18 cm height). Females individually housed in Macrolon plastic cages (MIII type, 18 cm height).
Lactation: Offspring were kept with the dam until termination.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF(R) SpezialdiätenGmbH, Soest, Germany) ad libitum
- Water (e.g. ad libitum): Tap-water ad libitum
- Acclimation period: 4 days priot to start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 07 Sep 2006 To: 03 Nov 2006

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

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 not showing evidence of mating were separated from their males

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On day 2 of the study, a formulation sample (1 mL) of each dose group was taken and stored at room temperature. The formulation and a sample of vehicle were dispatched to the test site for formulation analysis. Total aluminium concentrations were determined from samples containing nominal conentrations of 0, 8, 40 and 200 mg aluminium chlorid basic/mL. Total aluminium concentrations in these samples were <1, 677, 3674 and 18552 µg/mL, respectively. 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: 28 days
Females: 37-53 days

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- Only parental animals F0 were mated.
- Males were 9 weeks at mating.
- Females were 11 weeks at mating.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 40, 200, 1000 mg aluminium chloride basic/kg bw/day
Basis:
other: nominal in gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 3.6, 18, 90 mg Al/kg bw/day
Basis:
other: 9% aluminium (analytical concentration)

No. of animals per sex per dose:

40

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a 5-day range finding study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to start of treatment and at weekly intervals.


BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of exposure and weekly thereafter. Following evidence of mating, body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20, on lactation days 1 and 4, and at death.


FOOD CONSUMPTION: Yes
- Time schedule: weekly, for males and females. Not recorded during 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.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examinations
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, but water was provided
- How many animals: 5 males and 5 females randomly selected
- Parameters examined: White blood cells, differential leucocyte count, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin (concentration), platelets, prothrombin time, activated partialthromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examinations
- Animals fasted: Yes, but water was provided
- How many animals: 5 males and 5 females randomly selected
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The assigned males were tested during week 4 of treatment. The assigned females were tested during lactation.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity


GROSS PATHOLOGY: Yes. From 5 surviving animals/sex/group: adrenal glands, aorta, brain, caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymes, eyes, mammary gland area, femur, heart, ileum, jejunum, kidneys, larynx, lacrimal gland, liver, lung, lymph nodes (mandibular, mesenteris), nasopharynx, oesophagus, ovaries, pancreas, Peyer’s patches, pituitary gland, preputial gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions.
From all remaining animals: cervix, clitoral gland, coagulation gland, epididymes, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions.

HISTOPATHOLOGY: Yes. From 5 surviving animals/sex/group: adrenal glands, aorta, brain, caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymes, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (mandibular, mesenteris), oesophagus, ovaries, pancreas, Peyer’s patches, pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, vagina, all gross lesions.
From all remaining animals: cervix, clitoral gland, coagulation gland, epididymes, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions.


OTHER: Reproduction processes: Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.

Litter observations:

The numbers of live and dead pups at the First Litter Check (= check at day 1 of lactation) and daily thereafter (if possible, defects or cause of death were evaluated). 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 based on OECD guidance document on humane endpoints.
The individual weight of all live pups on days 1 and 4 of lactation.
Sex of all pups on days 1 and 4 of lactation (by assessment of the ano-genital distance)
The number of pups with physical or behavioural abnormalities daily.

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.

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 that 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 analyse the data:
- If the variables were assumed to follow a normal distribution, the Dunnett-test (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 was applied for pup organ weights.
- The Steel-test (many-to-one rank test) was applied if the data was not assumed to follow a normal distribution.
- The Fischer Exact-test 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 analyses were 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 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 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 x100).
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)

Details on results (P0)

Body weight
 
A slightly lower mean body weight was recorded for females at 90 mg Al/kg bw/d 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 occurrence.
Body weights and body weight gain of other treated animals remained in the same range as controls over the study period.
 
Haematology
 
The following minor, but statistically significant changes distinguished animals at 90 mg Al/kg bw/d from control animals:
-         lower mean corpuscular haemoglobin concentration in both sexes
-         higher platelet counts in males
Individual increases of haematology parameters at 90 mg Al/kg bw/d were not related to treatment-related morphological alterations. These changes included increased red cell distribution width and reticulocyte counts of two females, and increased relative eosinophilic counts of two males. Since a group response for these parameters was absent and the changes were of mild nature, no toxicological significance was ascribed to these alterations.
No dose-related response was apparent for the statistically lower haemoglobin levels of males at 3.6 mg Al/kg bw/d and higher, and for the changes in white blood cell counts of males and females at 3.6 mg Al/kg bw/d. 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 chemistry
 
The following statistically significant changes distinguished treated males from control animals:
-         lower alkaline phospahtase activity levels at 90 mg Al/kg bw/d
-         lower total protein levels at 90 mg Al/kg bw/d
-         lower albumin levels at 90 mg Al/kg bw/d
-         higher potassium levels at 50 and 90 mg Al/kg bw/d
-         higher inorganic phosphate at 90 mg Al/kg bw/d
There was, however, no or no evident dose-response relationship. Clinical chemistry parameters of females were similar to control levels.
 
Gross pathology
 
Red foci were noted on the glandular mucosa of the stomach of 5/10 males at 90 mg Al/kg bw/d, along with thickening of the glandular mucosa or limiting ridge in two of these cases.
No macroscopic abnormalities were noted among males at 18 mg Al/kg bw/d and females at 90 mg Al/kg bw/d.
 
Histopathology: non-neoplastic
 
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 90 mg Al/kg bw/d.

Reproduction parameters were unaffected by treatment up to 90 mg Al/kg bw/day. Mating performance, duration of gestation, fertility parameters, number of corpora lutea, number of implantation sites, and number of dead and living pups at first litter check were similar for the control and treated groups.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

18 mg/kg bw/day (nominal)

Based on:

other: total aluminium

Sex:

male/female

Basis for effect level:

other: gross pathology and histopathology: local stomach irritation effects

2

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

90 mg/kg bw/day (nominal)

Based on:

other: total aluminium

Sex:

male/female

Basis for effect level:

other: gross pathology and histopathology: local stomach irritation effects

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

90 mg/kg bw/day (nominal)

Based on:

other: total aluminium

Sex:

male/female

Basis for effect level:

other: lack of effects on reproductive, breeding and mating activity

Results: F1 generation

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 (3.6 mg Al/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
90 mg Al/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 90 mg Al/kg dose group of pups were observed.

3.6 and 18 mg Al/kg bw
No differences in the absolute and relative brain weight were observed between female pups born from dams exposed to 18 and 3.6 mg Al/kg of the Al chloride basic and female pups born from control females.


Gross pathology (offspring)

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)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

90 mg/kg bw/day (nominal)

Based on:

other: total aluminium

Sex:

male/female

Basis for effect level:

other: overall effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No adverse effects on reproductive behaviour, 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 (corresponding to 3.6, 18, 90 mg Al/kg bw/day) during pre-mating, gestation and short-time lactation period were reported. The NOAEL for reproductive toxicity (lack of reproductive /breeding, mating impairment and early postnatal developmental effects) was considered to be 1000 mg aluminium chloride basic/kg bw/day (90 mg Al/kg bw/day).
Inflammation of the glandular stomach mucosa was observed at 90 mg Al/kg bw/day (LOAEL local), but not at 18 mg Al/kg bw/day (NOAEL local) or below.