Aluminium trilactate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-27 to 2012-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium trilactate

EC Number:

242-670-9

EC Name:

Aluminium trilactate

Cas Number:

18917-91-4

Molecular formula:

C9H15AlO9

IUPAC Name:

Aluminium trilactate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Aluminiumtrilactat

Method

Target gene:

thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Basic medium: RPMI 1640 Hepes buffered medium + penicillin/streptomycin (50 U/mL and 50 μg/mL, 1 mM sodium pyruvate, 2 mM L-glutamin
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium)
Exposure medium:
-for 3 hour exposure: test substance in basic medium supplemented with 5% (v/v) heatinactivated horse serum (R5-medium)
-for 24 hour exposure: test substance in basic medium supplemented with 10% (v/v) heatinactivated horse serum (R10-medium)
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20), 5 μg/mL trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (R20)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, prior to experiment

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First experiment
Without S9-mix: 3, 10, 33, 100, 333, 1000, 1500, 2000 μg/mL
With 8% (v/v) S9-mix: 0.1, 1, 3.3, 10, 33, 100, 333 and 1000 μg/mL

Second experiment
Without S9-mix: 3, 10, 33, 100, 333, 750, 1000 μg/mL
With 12% (v/v) S9-mix: 0.1, 1, 3, 10, 33, 100, 333 and 1000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
- The final concentration of the solvent in the exposure medium was 0.8% (v/v)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: (without metabolic activation)

Remarks:

Migrated to IUCLID6: 15 µg/L (3 h), 5 μg/mL (24 h)

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: (with metabolic activation)

Remarks:

Migrated to IUCLID6: 7.5 - 10 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h/24 h in the absence of S9 mix (2 experiments), 3 h in the presence of S9 mix
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 12 d
- Fixation time (start of exposure up to fixation or harvest of cells): 15 d

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 independent experiments, 1 replicate each

NUMBER OF CELLS EVALUATED:
8E06 cells (1E06 cells/mL) for 3 hours treatment, 5E06 cells (1.25E05 cells/mL) for 24 hours treatment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (1E06) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 1E06 survivors and ≤ 170 per 1E06 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 106 survivors, and for CP not below 700 per 1E06 survivors.

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The pH and osmolarity of a concentration of 2942 μg/mL were 5.32 and 0.408 Osm/kg respectively
(compared to 7.33 and 0.424 Osm/kg in the solvent control).

Precipitation:
First experiment: After 15 minutes, the test substance formed a heavy suspension at the dose level of 1000 μg/mL.
In the second experiment the test substance precipitated in the exposure medium at concentrations of 100 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance
concentration range of 33 to 2942 μg/mL in the absence of S9-mix with a 3 and 24 hour treatment
period and in the presence of S9-mix with a 3 hour treatment period. The relative suspension growth was 37 and 42% at the highest test substance concentration of 2942 μg/mL compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively. In the absence of S9-mix, the relative suspension growth was 7% at the highest test substance concentration of 2942 μg/mL compared to the relative suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the
minimum and maximum value of the historical control data range. Except in the first experiment, in which the mutation frequency of one of the solvent control cultures in the absence of S9-mix was just below the historical control data range.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Aluminium trilactate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this study.

Executive summary:

In a mammalian cell gene mutation assay according to OECD guideline 467, adopted July 21, 1997 (thymidine kinase (TK)), L5178Y mouse lymphoma cells cultured in vitro were exposed to Aluminium trilactate (93.1% purity) in DMSO in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

 

First experiment

Without S9-mix: 3, 10, 33, 100, 333, 1000, 1500, 2000μg/mL

With 8% (v/v) S9-mix: 0.1, 1, 3.3, 10, 33, 100, 333 and 1000μg/mL

 

Second experiment

Without S9-mix: 3, 10, 33, 100, 333, 750, 1000μg/mL

With 12% (v/v) S9-mix: 0.1, 1, 3, 10, 33, 100, 333 and 1000μg/mL

 

Aluminium trilactate was tested up to cyctotoxic concentrations. The positive controls induced the appropriate response. There was noevidenceof induced mutant colonies over background.

 

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.