Aluminium triformate

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:

27 January 2010 to ...

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP- and guideline compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium triformate

EC Number:

230-898-1

EC Name:

Aluminium triformate

Cas Number:

7360-53-4

Molecular formula:

CH2O2.1/3Al

IUPAC Name:

aluminium triformate

Details on test material:

Identity: Aluminium triformate
Batch No.: 128110/004
Molecular Weight: 216.1 g/mol (with crystal water) 162.0 g/mol (without crystal water)
Purity: 97.3 %
Stability in Solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: Not stated

Method

Target gene:

autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
hypoxanthine 1.0x10-4 M
aminopterin 2.0x10-7 M
thymidine 1.6x10-5 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
hypoxanthine 1.0x10-4 M
thymidine 1.6x10-5 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

First experiment (doses applied, but not evaluated: in parentheses), without and with metabolic activation, 4 h treatment:
(17.5), 35.0, 70.0, 140.0, 280 and 560 µg/mL
Second experiment (doses applied, but not evaluated: in parentheses), without metabolic activation, 24 h treatment:
(17.5), 35.0, 70.0, 140.0, 280 and 560 µg/mL
Second experiment (doses applied, but not evaluated: in parentheses), with metabolic activation, 4 h treatment:
(17.5), 35.0, 70.0, 140.0, 280 and 560 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: On the day of the experiment (immediately before treatment), the test item was suspended in deionised water. The final concentration of deionised water in the culture medium was 10 % v/v. The test item concentrations was corrected for the purity of the test item (97.23 %).

- Justification for choice of solvent/vehicle: comon vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: (cultures without metabolic activation, Sigma, 82024 Taufkirchen, Germany, lot 76296KJ; expiry date May 2010; purity > 99%; final Concentration: 19.5 µg/mL = 0.18 mM (experiment I) and13.0 µg/mL = 0.12 mM (experiment II) )

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with metabolic activation; Aldrich Chemie, 89555 Steinheim, Germany; batch 0971311; expiration date March 2011; purity: ≥ 98 %; final concentrations: 3.0 µg/mL = 10.7 µM and 4.5 µg/mL = 16.1 µM )

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: yes
- Exposure duration: 4 hours (first experiment with/without metabolic activation and second experiment with metabolic activation) and 24 hours (second experiment without metabolic activation)
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: The survival rate and viability were determined based on the Poisson distribution method. The zero term of the Poisson distribution, [P(0)] method, was used.

Evaluation criteria:

Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

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
- Effects of pH and osmolality: The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation:
Solvent control: pH 7.46, osmolarity 255 mOsm
Aluminium triformate, 2220 μg/mL: pH 7.43 (adjusted with 70 μL 2N NaOH), osmolarity 291 mOsm
- Precipitation: Precipitation of the test item visible to the naked eye was noted at 280 μg/mL and above in both main experiments with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: yes, see above

COMPARISON WITH HISTORICAL CONTROL DATA: yes, see also attachment

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For tables see Attachment.

No relevant toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

The isolated minor reduction of the relative total growth to 46.1 % in the first culture of the second experiment with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions. The reduction is most likely based on precipitation of the test item leading to unspecific toxic effects by mechanical shear forces.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments up to the maximum acceptable concentration with and without metabolic activation at both treatment intervals. In the first experiment the historical range of solvent controls was exceeded in culture II without metabolic activation at 140 μg/mL. However, the threshold of 126 above the mutation frequency of the corresponding solvent control was not reached by far and no comparable increase was noted in the parallel culture under identical conditions. In the second experiment the historical solvent control data were also exceeded in culture I without metabolic activation at the concentrations of 35.0 to 280 μg/mL and at the maximum concentration of 560 μg/mL with metabolic activation. Again, these effects were not reproduced in the parallel culture II performed under identical conditions.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second experiment in the second culture without metabolic activation and in the first culture with metabolic activation. Since the mutation frequency did not exceed the threshold as indicated above, the statistical result is considered as biologically irrelevant fluctuation.

In this study the range of the solvent controls was from 93 up to 200 mutant colonies per 106 cells; the range of the groups treated with the test item was from 85 up to 299 mutant colonies per 106 cells. The highest solvent control values (200, 198, and 190 colonies per 106 cells) exceeded the recommended 50 – 170 x 106 control range as stated under paragraph 8.12, acceptability of the assay of this report. The data are judged as acceptable however, since the solvent controls remained within the range of 50-200 colonies per 106 cells, originally recommended by the IWGT (11). The cloning efficiency exceeded the upper limit of 120% in the second culture of the second experiment without metabolic activation (140%). The data are acceptable however, since the cloning efficiency of the parallel culture remained within the acceptable range. Cloning efficiency values above 100% occasionally occur since even suspension cell cultures do not form an ideal solution in medium. The cells tend to form transient aggregates that are counted as single cells during determination of the cell density. The aggregation does not compromise the validity of the data however, since the absolute values of the cloning efficiency are used to calculate the mutation frequency.

MMS (19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II) and CPA (3.0 μg/mL and 4.5 μg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity at at least one of the concentrations of the controls.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of Aluminium triformate to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest concentration (2220 μg/mL) of the pre-experiment was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

The dose range of the experiments was adjusted to toxicity data.

Conclusion

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, Aluminium triformate is considered to be non-mutagenic in this mouse lymphoma assay.