Aluminium ammonium bis(sulphate)

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:

11 September 2006 - 03 November 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study conformed to an international screening test guideline (OECD 422), was performed according to Good Laboratory Practice . Data from Aluminium chloride are relevant for ammonium alum considering the dissociation of both salts as aluminium ions (and chloride or sulfate and ammonium ions) after oral administration.

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

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:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

basic Aluminum chloride

IUPAC Name:

basic Aluminum chloride

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

Constituent 3

Reference substance name:

Aluminium chloride, basic

IUPAC Name:

Aluminium chloride, basic

Details on test material:

- Name of test material (as cited in study report): Aluminium chloride basic
- Substance type: Aqueous solution
- Physical state: Liquid
- Analytical purity: No data
- Impurities (identity and concentrations): Confidential information
- Composition of test material, percentage of components: Confidential information
- Lot/batch No.: Confidential information
- Expiration date of the lot/batch: Confidential information
- Stability under test conditions: 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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation:
Males: approx. 9 weeks
Females: approx. 11 weeks
- Weight at study initiation:
Males: mean 266-269 g, SD 6.6-12.2
Females: mean 237-240, SD 7.0-10.7
- Housing: According to guideline
Pre-mating: groups of 5 animals/sex/cage
Mating: 1 male, 1 female
Post-mating: males in groups of 5 animals/sex/cage, females individually
Lactation: offspring together with dam
- Diet (e.g. ad libitum): Ad libitum, pelleted rodent diet
- Water (e.g. ad libitum): Ad libitum, tap-water
- Acclimation period: 4 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-23.4
- Humidity (%): 37-95 (guideline: max. 70%, but no effects expected)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not relevant

Vehicle:

other: Water (Milli-U)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
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.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: ?
- Amount of vehicle (if gavage): 5 ml/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: Not relevant

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: 28 days (14 days prior to and 14 days during mating)
Females: 28 days (14 days prior to and 14 days during mating) + 9-25 days (during gestation and lactation)

Frequency of treatment:

Once daily, 7 days a week

Details on study schedule:

Not relevant

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
40, 200, 1000 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
3.6,18,90 mg Al 3+/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 dose range finding study (NOTOX project 473524)
- Rationale for animal assignment (if not random): Random
- Section schedule rationale (if not random): No data

Positive control:

Not relevant

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations checked: Mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: At study start, once weekly and at death (females: gestation days 0, 4,7, 11, 17, 20, lactation day 1, 4)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

WATER CONSUMPTION: Subjective appraisal but not recorded

OTHER:
FUNCTIONAL OBSERVATIONS:
Males during week 4, females during lactation; 5 M and5F, randomly selected from all groups:
Hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity test (recorded 12 hrs overnight)

REPRODUCTIVE BEHAVIOUR:
Male number paired with, mating data, confirmation of pregnancy, delivery day

Oestrous cyclicity (parental animals):

Not performed

Sperm parameters (parental animals):

No data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Not applicable

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
numbers of live and dead pups( daily)
individual bw on day 1 and 4 of lactation
sex of all pups on day 1 and day 4 by assessment of ano-genital distance
physical or behavioural abnormalities daily

GROSS EXAMINATION OF DEAD PUPS:
Yes, sexed and externally examined if practically possible. The stomach was examined for the presence of milk.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 29 of study
- Maternal animals: All surviving animals on day 4 post partum

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination of all organs of all animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] (see "Any other information on materials and methods incl. tables) were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
The F1 offspring was sacrificed at 4 days of age.

GROSS NECROPSY
All offspring sexed and externally examined. Stomach examined for the presence of milk
All macroscopic abnormalities recorded, and defects or cause of death evaluated. Any abnormal pup preserved in 10% buffered formaldehyde

Statistics:

Dunnet -Test (Dunnet, 1955) based on pooled variable estimate for variables assumed to have a normal distribution
Sett-test (Miller, 1981) for variables asssumed not to follow a normal distribution
Student’s T- test for pup organ weights
Fisher exact test for frequency data. All tests two-sided, P<0.05

Reproductive indices:

Percentage mating
Fertility index
Conception rate
Gestation index

Offspring viability indices:

Percentage live male & female pups
Percentage post-natal loss
Viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Observations: Mortality / viability at least twice daily Detailed clinical signs at least once daily Body weights at start, once weekly and at death (females: gestation days 0, 4,7, 11, 17, 20, lactation day 1, 4 food consumtion weekly water consumption

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See RS on 28-day repeated dose part of study

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See RS on 28-day repeated dose part of study

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histopatological abnormalities were obsurved

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Dose volume 5 ml/kg bw. 0, 40, 200, 1000 mg/kg/day of the test substance solution, equal to 0, 3.6, 18, 90 mg/kg bw/ d of Al3+

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

M/F ratio : 1/1 per cage Maximum of 14 days, separation once mating occurred (i.e evidence of sperm in vaginal lavage or intravaginal copulatory plug) Proof of pregnancy: littering and post-mortem examination of reproductive organs

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

% pregnant per dose level: see under details on results

Details on results (P0)

For Food consumption , Clinical signs, Haematological findings and duration, Clinical Biochemistry findings, Gross pathology : See RS on 28-day repeated dose part of study


Body weight (parenteral animals)
Slightly lower mean bw for females at 1000 mg/kg bw/d (90 mg/kg bw/d) at day 8 with slight weight loss for three of these females. BW recovered during the following mating and post-coital period. In the other groups no deviations compared to controls

Parameters assessment during study (maternal and fetal)
Clinical signs as above
Percentage mating
Fertility index
Conception rate
Gestation index
Percentage live male & female pups
Percentage post-natal loss
Viability index

% pregnant per dose level:
Dose (mg/kg bw/d) 0 40 200 1000
% pregnant 100 90 90 90

Number aborting:
None

Number of resorptions, early/late if available:
Number of implantations:
Pre and post implantation loss, if available; Number of corpora lutea
Corpora lutea:
no significant differences between exposed and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Corpora lutea 18.5 (2.01) 18.2 (7.94) 15.0 (5.40) 16.9 (1.91)
Implantation sites:
no significant differences between exposed and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Inplantation sitea 16.6 (2.55) 15.7 (5.72) 14.0 (5.35); 16.2 (1.99)
Litter size:
no significant differences between exposed and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Litter size 15.1 (3.1) 14.0 (5.1) 14.7
(2.7) 14.8
(1.9)

Duration of Pregnancy:
No difference between dose groups and controls
Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
MF ratio 21.9 (0.3) 21.9
(0.6) 21.9
(0.3) 21.8
(0.4)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: No abnormalities at any dose level

2

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

Aluminium (Al3+)

Sex:

female

Basis for effect level:

other: No abnormalities at any dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No difference between exposed and controls Alive / dead (nrs per group): Dose (mg/kg bw/d) 0 40 200 1000 Live /dead 136/0 126/12 132/1 148/0

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Litter size: no significant differences between exposed and controls Mean (SD): Dose (mg/kg bw/d) 0 40 200 1000 Litter size 15.1 (3.1) 14.0 (5.1) 14.7 (2.7) 14.8 (1.9) Litter weights: No difference between exposed and controls Lactation day 1; M

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

Grossly visible abnormalities, external, soft tissue and skeletal abnormalities : No abnormalities observed either in controls or in exposed animals ouside the normal biological variation for race/strain No behavioural abnormalities in pups observed

Histopathological findings:

not examined

Details on results (F1)

Sex ratio
No difference between exposed and controls
M / F ratio:
Dose (mg/kg bw/d) 0 40 200 1000
MF ratio 1.1 0.84 0.99 0.92


Postnatal growth
No difference between exposed and controls
Postnatal weight, Lactation day 4; Mean (SD):
Dose (mg/kg bw/d) 0 40 200 1000
Weight (gr) 10.5 (1.5) 10.3 (0.9) 10.5 (1.4) 10.4 (0.6)

Postnatal survival
No difference between exposed and controls
Post-natal pup deaths:
Dose (mg/kg bw /d 0 40 200 1000
Nr. Of dead pups 1 2 0 3

Organs examined at necropsy (macroscopic and microscopic)
Pups:
All offspring sexed and externally examined. Stomach examined for the presence of milk
All macroscopic abnormalities recorded, and defects or cause of death evaluated. Any abnormal pup preserved in 10% buffered formaldehyde

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No abnormalities at any dose level

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

90 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

Aluminium (Al3+)

Sex:

male/female

Basis for effect level:

other: No abnormalities at any dose level

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

1 female of the control group sacrified in extremis at day 22 of pregnancy due to parturition difficulties

Applicant's summary and conclusion

Conclusions:

This study revealed no maternal toxicity at any dose and no reproductive, breeding or developmental toxicity at any dose from two weeks prior to mating to at least 3 days of lactation (females). Therefore, a NOAEL for maternal local and systemic toxicity of 1000 mg/kg bw/d was established. For reproductive and developmental toxicity the NOAEL was 1000 mg/kg bw /d (equivalent to 90 mg Al3+/kg bw/d Al).

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 bw corresponding to 3.6, 18 and 90 mg Al³⁺/kg bw/d.

Treatment with Al chloride basic by oral gavage revealed paternal toxicity (irritation effect on glandular stomach mucosa, local effect) at 1000 mg/kg bw/d 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 bw/d and LOAEL – at level 1000 mg/kg bw/d, for both males and females.

Several statistically significant changes in clinical biochemistry parameters were observed at 1000 mg/kg bw/dsuggesting 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 baseline values for the observed clinical data limit the interpretation of the results. The authors consider the 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 bw/d 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/d (i.e. 90 mg Al³⁺ /kg bw/d).