Pyridinium, 1-butyl-, .mu.-chlorohexachlorodialuminate(1-) (1:1)

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: oral

Remarks:

other: developmental neurotoxicity and chronic toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Double-blind, vehicle-controlled randomized twelve-month neurodevelopmental toxicity study of common Aluminium salts in the rat.

Author:

Poirier J., Semple H., Davies J., Lapointe R., Dziwenka M., Hiltz M., Mujibi D.

Year:

2011

Bibliographic source:

Neuroscience. 193: 338-362.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 426 and OECD 452

Deviations:

yes

Remarks:

food consumption is missing; exposure during in utero (GD 6-21) and weaning period (post-natal day (PND) 1-21), but the exposure of the rats to Al citrate continued beyond this period, until 12 months of age

Principles of method if other than guideline:

Timed pregnant dams (n= 20 per group) were administered with aqueous solutions of aluminium citrate at 3 dosage levels (30, 100 and 300 mg Al/kg bw/day (nominal). Two control groups received either a sodium citrate solution (citrate control with 27.2 g/L of citrate) or plain water (control group).
Al citrate was administered to dams via drinking water ad libitum from gestation day 6 through weaning of offspring. Litter sizes were normalized (4 males and 4 females) at postnatal day 4. Offspring (female and male) were used to assess behavioral ontogeny, cognitive function, brain weight, clinical chemistry, hematology, tissue/blood levels of aluminium and neuropathology at various doses (30, 100 and 300 mg Al/kg) and time points (at PND 23 and 64, 120 and 364).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium citrate

EC Number:

250-484-4

EC Name:

Aluminium citrate

Cas Number:

31142-56-0

IUPAC Name:

aluminum citrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Aluminium Citrate
- Supplier: Chemos GmbH (Regenstauf, Germany)
- Appearance: white powder
- Purity: 9.3% , 9.8% and, 8.7% Aluminium by mass as per the certificates of analysis from the supplier for each lot.
- Storage: Room Temperature
- pH adjusted between 6 and 7

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River
Acclimatization: 9 days
Allocation: random
Diet: Rats were fed AIN-93G growth food (Purina TestDiet) until PND 95–99, followed by AIN-93M maintenance food (Purina TestDiet) for the duration of the study
Housing: singly in shoebox or ventilated cages except during breeding (wire-bottomed cages) and until weaning (grouped with rest of litter) for the pups
nly animals in apparent good health and within the specified age range were selected for randomization to treatment groups.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

other: deionized water

Details on exposure:

The dose materials (aluminum citrate) were calculated and prepared from three different lots from Chemos GmbH, Regenstauf, Germany. The aluminum content by mass was 9.3%, 9.8% and 8.7% respectively based on the manufacturer product sheet. Fresh filtered solutions were prepared weekly using de-ionized water and the pH was adjusted between 6 and 7. The final concentrations of Al for each of the doses have been independently assessed using inductively coupled plasma mass spectrometric analyses of metal concentrations by Maxxam Analytics Inc. (Burnaby, Canada) using certified GLP. Positive and negative control solutions: the negative control solution consisted of de-ionized water, whereas the positive control solution (placebo) consisted of sodium citrate at a concentration of 27.2 g/L. The latter concentration mimics the citrate ionic strength of the highest aluminum citrate solution.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Inductively coupled plasma mass spectrometric analyses of metal concentrations in aluminum citrate and sodium citrate powders, in rat diet food, in deionized water, in aluminum dosing solutions, and in blood, brain, liver, kidney, bone and spinal cord tissues was conducted by Maxxam Analytics Inc. (Burnaby, Canada) using certified GLP. Specifically, aluminum (Al), iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn) were quantified using a Perkin Elmer ELAN 6000 ICP-MS apparatus according to the manufacturer's standardized GLP protocols.

Duration of treatment / exposure:

On gestational day 6, the test item was administered to groups of pregnant animals during gestation, lactation, and to offspring during post-weaning, through to post-natal day 364 for cohort 4.

Dams
GD 6 to PND 21.

Pups (males and females)
PND 22 to PND 364.

Cohort 1 – GD 6-21, PND 1-22
Cohort 2 – GD 6-21, PND 1-64
Cohort 3 – GD 6-21, PND 1-120
Cohort 4 – GD 6-21, PND 1-364

Frequency of treatment:

daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
30, 100, 300 mg Al /kg bw
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 27.2 g sodium citrate / L
Basis:
analytical conc.

No. of animals per sex per dose:

Dams: 20/group;
Offspring: 10-20 females and 10-20 males/group;
Litters: 20 litter/dose.

Control animals:

yes, concurrent vehicle

other: sodium citrate @ equimolar Al concentration (27.2 g/L) as compared to high dose group (300 mg Al / kg bw)

Details on study design:

Blinding
Treatment groups were identified with letters A–E. Test facility staff were blinded as to which letter (A–E) denoted which of the treatment groups (1–5. Blinding was broken once draft contributing scientist reports had been acquired from the principal investigator (metals analysis), the statistician and the pathologist.


Breeding, gestation, parturition, and lactation
Animals were segregated into breeding pairs and allowed the opportunity to breed for up to five consecutive nights. During the breeding period, female animals were checked daily for the presence of vaginal plugs (indicating insemination). The date of breeding (i.e. insemination) was defined as the day when a vaginal plug was first detected. Dams consumed the dosing solution throughout gestation and lactation. Dams were observed, weighed and fluid consumption was taken. On PND 1, dams and litters were moved to their designated rooms and on the culling day (PND 4–8), were allocated to rack and position using a Youden square so that there was approximately equal representation of groups within each shelf of the racks. Four days after birth, beyond normalization to four males and four females per litter, pups were assigned to each of four sacrifice day groups associated with milestone observations and sacrifice. Litters were further allocated to subgroups for regular necropsy (A) and perfusion fixation (B). Because the litters were already randomized, every second litter was assigned to the perfusion fixation subgroup. Pups were weighed. Fluid consumption was monitored. Daily observations were made on all pups. Pups were weaned at PND 22 by moving them to individual ventilated caging and another Youden square was used to determine distribution of the pups within the rack.

Examinations

Observations and clinical examinations performed and frequency:

All dams underwent daily morbidity and mortality checks while in the study, and underwent Functional Observational Battery (FOB) examinations on gestational days 7 and 13, a clinical examination on the day of delivery, and FOB examinations on PNDs 3 and 10.
Unless undergoing clinical examination on a given day, all pups underwent morbidity and mortality checks on a daily basis from PND 1 through PND 366. Pups from the Na citrate and high-dose groups received clinical observations at least weekly from PND 33 to PND 153. Prior to PND 4, morbidity and mortality checks were recorded on the dam's observation form. On and after PND 4, pups in the normalized litters had observations recorded on their own forms. Additional neurobehavioral observations (FOB, T-maze, Morris Water Maze, Auditory Startle and Motor Activity) were carried out at specified intervals.

Specific biochemical examinations:

The biochemical parameters were evaluated on a Hitachi 912 Automatic Analyzer using blood collected under isoflurane anesthesia on the day of scheduled sacrifice, prior to euthanasia, preferably via venipuncture of the abdominal vena cava. List of biomarkers examined for each animal can be found at http://download2.aitf.ca/admin-7898196ecbf1c96e6894b78433ef56a2/AluminumStudyAppendices.zip.

Neurobehavioural examinations performed and frequency:

-- FOB
One male and one female pup from each litter (pups 4 and 8, which were allocated to the day 364 group) underwent FOB examinations on PNDs 5, 11, 22, 36, 45, 56, and biweekly thereafter until the week of PND 350.

-- T-maze
T-maze tests were conducted on PND 22. A T-maze test of memory and spatial recognition in weanling rat pups was conducted according to (Crofton et al., 1993). The specific test used was a spontaneous alternation test with no reinforcement, in which the animal's propensity to explore new areas was observed and measured. The T-maze for rats was from San Diego Instruments (San Diego, CA, USA). Visual cues were present on the inside walls of the T maze (stripes, geometrical shapes) to assist the subject with orientation. By plotting the number of errors over time, a learning curve is generated for each rats.

Morris water maze
The Morris water maze tests were conducted prior to PNDs 59, 61, 63 and 64, PNDs 114, 116, 118 and 119, and PNDs 357, 359, 361 and 362 as described before (Champagne et al., 2002) (see Table 3). The in-life portion of the study consisted of three blocks of four trials for a total of 12 training trials which were conducted over three alternate testing days (e.g. Thursday, Saturday and Monday), one day for each block, followed by a block of probe trials (two platform-removed probe trials and two visible platform trials) conducted the day after the last of the three blocks (e.g. Tuesday) and all animals from the scheduled investigations group for that date were tested on all four days for Day 64, 120 and 364 groups. The trials were intended to be run at the same time of day (i.e. ±2 h) for each animal, however due to scheduling conflicts, some trials were run outside of this range for Day 64, 120 and 364 groups. The order of the animals from the 10 groups (five treatments×2 sexes) being tested was to be randomized and counterbalanced to minimize the effect of time of day when testing was done. The animals were already randomized to rack placement and therefore they were tested in rack order. This met the requirement for randomization and counterbalancing.

Statistics:

Statistical analyses were conducted using SAS® Release 9.1 for Windows. Data collected on dams and pups was analyzed separately and all statistical analysis on the pups was performed separately for each sex. Analyses for pups were done separately for each test day group unless specified otherwise. Statistical significance was declared when P≤0.05. All statistical analyses were done based on the principle of intention-to-treat.