Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
5 Jan 2010 – 4 Feb 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): Al(OH)3 /SH-20 Muster
Source: No information (sponsor)
Manufacture date: week 40, 2009
Expire date: 25 years from manufacture
Batch number: 40/2009
Received: December 2, 2009
Storage: at 15-25 °C in the dark
Physical form: white powder
Purity: 99%
Moisture: 0.03±0.02%
Impurities:
Analysis in dried material at 105 °C:
Al2O3 – 65.1±0.3%
SiO2 – 0.007%
Fe2O3 – 0.006%
TiO2 – 0.001%
CaO – 0.003%
Na2Oges – 0.18%
V2O5 - <0.001%
P2O5 - <0.001%
ZnO - <0.001%
Ga2O3 – 0.005%
Granule distribution <45 µm – 4.7±1%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Age at study initiation: range-finder experiment: 6-10 weeks; micronucleus experiment: 8 weeks
- Weight at study initiation: range-finder experiment: 181-190 g; micronucleus experiment: 217-260 g
- Housing: The rats were housed in an air-conditioned room (15 air exchanges/hour) in groups, up to six per group, “in cages with the 'Code of practice for the housing and care of animals used in scientific procedures”
- Diet (e.g. ad libitum): ad libitum access to SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham)
- Water (e.g. ad libitum): Mains water ad libitum via water bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): fluorescent lighting, 12/12-h light/dark cycle (light from 06:00 to 18:00h)

- Identification: individually, by uniquely numbered ear-tag.
Cages were identified by study number, study type, start date, number and sex of animals, dose level and proposed time of necropsy using a color-coded procedure

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
1% Carboxymethylcellulose in deionised water (1% CMC)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Freshly prepared. As no storage instructions were available, after preparation the formulations were held at 15-25 °C in a dark place and used within 2 hours.

Formulations were mixed using a Silverson homogenizer until visibly homogenous; dose bottles were stirred continuously on a magnetic stirrer before and throughout dosing


Duration of treatment / exposure:
not applicable
Frequency of treatment:
Two doses ≈ 24 hours apart
Post exposure period:
24 hours after the second (final) administration
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
actual ingested
Range-finder experiment: 2000 mg/kg/day and Micronucleus experiment: 500, 1000 and 2000 mg/kg/day
No. of animals per sex per dose:
Range-finder experiment – 6 (3 males and 3 females)
Micronucleus experiment – 6 (males only)
Control animals:
yes, concurrent vehicle
Positive control(s):
Substance: Cyclophosphamide (CPA), Sigma-Aldrich Chemical Co, Poole, UK, freshly prepared in saline
Administration: once via oral gavage 24 hours prior to necropsy (dose 20 mg/kg)

Examinations

Tissues and cell types examined:
Bone marrow cells were obtained from the femur.
Details of tissue and slide preparation:
Measurement of study outcomes
Bone marrow cells were obtained from the femur. Slides were stained with acridine orange and scored using fluorescence microscopy.

“Slides from the CPA-treated rats were initially checked to ensure the system was operating satisfactorily.”

Slides from all groups were arranged by randomly allocated animal number and analyzed by an individual unaware of the animals’ dose group.

The relative proportion of polychromatic erythrocytes (%PCE) was determined by analyzing at least 1000 cells - polychromatic plus normochromatic erythrocytes (NCE)

Frequency of micronucleated PCE (% MN PCE) was determined by analysis for micronuclei (MN) of at least 2000 PCE per animal.

The following data are presented in a tabular form for each animal: PCE and NCE counts; %PCE; micronucleated PCE (MN PCE) per 2000 PCE; %MN PCE

For each group, the following values are presented: cell total, %PCE, total MN PCE, mean MN PCE per 2000 PCE, % MN PCE (SD)

The laboratory historical vehicle control ranges are presented and the MN PCE data from the vehicle control group are compared with these historical data

The laboratory historical positive control ranges are also presented

Ancillary endpoints examined (e.g. general toxicity):
Routine health status checks – at the beginning and the end of each work day.
Range-finder experiment:
- clinical signs of toxicity (immediately after each dose administration, at least 4 times during the four-hour post-administration period and prior to the second dose),
- body weight (each day of dosing and each day post-administration)
- core body temperature (once in the 24 hours pre-administration, 2 and 4 hours after each administration and once on the first post-administration day)
Micronucleus experiment:
- clinical signs of toxicity (immediately after each dose administration, at least 4 times during the 4-hour post-administration period, prior to the second dose and on the day of bone marrow sampling)
- body weight - on the day of bone marrow sampling
- as no changes in body temperature were observed in the range-finder experiment, body temperature was not measured.
Evaluation criteria:
The criteria used for a positive response are provided explicitly.
For the test article to be considered positive (inducing clastogenic/aneugenic damage), all of the following 4 criteria are to be met:
“1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. The group mean MN PCE value at such a point exceeds the 95% calculated confidence interval for the mean historical vehicle control data
4. A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).”
If none of the 4 criteria are met, the test article is to be considered negative in this assay.
Results only partially satisfying the above criteria are to be considered on a case-by-case basis. Biological relevance is to be taken into account (e.g. consistency of response within and between dose levels)
Statistics:
Heterogeneity chi-square test was used for evaluation of inter-individual variation in the numbers of MN PCE for each group.

A 2x2 contingency table and chi-square test was used to compare the numbers of MN PCE in each treated group with the numbers in vehicle control groups

A test for linear trend was used to evaluate possible dose-response relationship.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

OECD TG #474: Principal endpoint = Frequency of micronucleated immature (polychromatic) erythrocytes

 

1. The frequency and distribution of MN PCE in the vehicle control group weresimilar to the historical vehicle control data.

2. There was a significant increase in the frequency of MN PCE (% MN PCE) in the positive control group

3. There was no evidence of test-substance-induced bone marrow toxicity, i.e. no decrease in the relative proportions of PCE (%PCE) compared to the vehicle control group and no dose-dependent decrease in %PCE; %PCE in the treated groups were even slightly higher than in the non-treated groups.

4. Group mean frequencies of MN PCE (% MN PCE) in all three dose groups were similar to and not significantly different from those in the vehicle control group.

5. Individual %MN PCE for all treated animals were within the range of historical vehicle control distribution data and similar to those observed in recent historical controls.

------------------------------------------------------------------------

Group      %PCE   MN PCE/2000 PCE    %MN PCE (SD)

(dose)                                                           

-------------------------------------------------------------------------

0              49.35             2.67                   0.13 (0.10)

500           58.55             2.33                   0.12 (0.09)

1000         53.08             2.83                   0.14 (0.09)

2000         54.82             2.50                   0.13 (0.06)

PC*          46.13            55.50                  2.78 (1.60)

-------------------------------------------------------------------------

*PC-positive control

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Aluminium hydroxide adminaterede“It is concluded that aluminium hydroxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 2000 mg/kg/day (the maximum recommended dose for this study)”
Executive summary:

Covance (2010a) administered aluminium hydroxide to out-bred male Sprague Dawley rats to examine the induction of micronuclei (MN) in bone marrow polychromatic erythrocytes (PCE). The animals were randomized into 5 groups (6 animals in each). Three groups were exposed to doses of 500, 1000, and 2000 mg/kg/day, one group (negative control) received the vehicle (1% carboxymethylcellulose in deionised water), and one group (positive control) received a known mutagen, Cyclophosphamide. The test substance was administered by oral gavage in two doses 24 hours apart. The maximum dose tested was selected based on data from a range-finder experiment. The principal endpoint was the frequency of micronucleated PCE (% MN PCE) in the bone marrow, sampled 24 hours after the final test substance administration. The results of the study were negative: group mean % MN-PCE values in all three dose groups were not significantly different from those in the vehicle control group; individual %MN PCE for all treated animals were also within the range of historical vehicle control distribution data.

No signs of general toxicity or bone marrow toxicity (based on the proportions of immature erythrocytes) were observed in this study. The authors concluded: “……aluminium hydroxide did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 2000 mg/kg/day.” This GLP-compliant study was conducted in accordance with OECD Test Guideline #474 (1997) and European Agency for the Evaluation of Medicinal Products (1995) guidelines. Small deviations were unlikely to impact the validity of the results. A Klimisch Score of 1 was assigned to this study. The MN assay results are reliable but require discussion in the context of toxicokinetic information as Al levels were not determined in the target tissues.