Fatty acids, C16-18 (even numbered), aluminum salts

Genetic toxicity: in vivo

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:

key study

Study period:

5 Jan - 4 Feb 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-Guideline study, tested with the source substance aluminium hydroxide. According to the ECHA guidance document "Practical guide 6: How to prepare read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline (European Agency for the Evaluation of Medicinal Products. (1995) ICH Topic S 2 A. “Genotoxicity: Guidance on Specific Aspects of Genotoxicity Tests for Pharmaceuticals”).

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium hydroxide

EC Number:

244-492-7

EC Name:

Aluminium hydroxide

Cas Number:

21645-51-2

Molecular formula:

AlH3O3

IUPAC Name:

aluminum trihydroxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Al(OH)3 /SH-20 Muster
- Physical state: white powder
- Analytical purity: 99%
- Composition of test material, percentage of components: 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%
- Lot/batch No.: 40/2009
- Expiration date of the lot/batch: week 40, 2034
- Storage condition of test material: at 15-25 °C in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Age at study initiation: range-finding study: 6-10 weeks; main experiment: 8 weeks
- Weight at study initiation: range-finding study: 181-190 g; main experiment: 217-260 g
- Assigned to test groups randomly: yes (main experiment)
- Housing: up to 6 animals per cage
- Diet: SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham), ad libitum
- Water: mains water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: carboxymethylcellulose in deionised water (1% CMC)
- Justification for choice of solvent/vehicle: CMC is a well-known vehicle, and it was tested previously in the test laboratory
-- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were freshly prepared by mixing with a Silverson homogenizer until visibly homogenous. Dose bottles were stirred continuously on a magnetic stirrer before and throughout dosing. The dosing solutions were used within 2 h.

Duration of treatment / exposure:

2 days

Frequency of treatment:

daily

Post exposure period:

24 h after treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

range-finding study: 3 (males and females)
main experiment: 6 (male)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): due to the known properties of inducing micronuclei formation, cyclophosphamid was selected as appropriate positive control.
- Route of administration: oral (24 h prior to necropsy)
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range-finding study performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): One bone marrow sample was taken 24 h after the final treatment.

DETAILS OF SLIDE PREPARATION: Slides were stained with acridine orange and scored using fluorescence microscopy.

METHOD OF ANALYSIS: Slides from all groups were arranged by randomly allocated animal number and analyzed by an individual unaware of the animals’ dose group. Scoring was therefore blinded.

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.

Evaluation criteria:

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 examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No clinical signs of toxicity, nor effects on body weight or core temperature were observed in the range-finding study.


RESULTS OF DEFINITIVE STUDY
1. The frequency and distribution of MN PCE in the vehicle control group were similar 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.


General toxicity:
No clinical signs of toxicity were observed in any animal in any group (vehicle control, positive control or any dose group)
Body weight:
No effect was observed on body weight.

Any other information on results incl. tables

Table 1: Results after exposure to aluminium hydroxide

Group      %PCE   MN PCE/2000 PCE    %MN PCE

(dose)                                                            (SD)

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

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)

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*PC-positive control

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative