Lanthanum trinitrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

From 30 August 2012 to 14 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented GLP study performed with lanthanum acetate according to OECD Guideline 473. We refer to the read across justification added in section 13 to the IUCLID file for extrapolation to lanthanum trinitrate. A maximal reliability score of 2 (reliable with restrictions) is assigned because the study has been used for read-across purposes in this dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

100587-90-4

Cas Number:

100587-90-4

IUPAC Name:

100587-90-4

Constituent 2

Reference substance name:

Lanthanum (3+) acetate

IUPAC Name:

Lanthanum (3+) acetate

Details on test material:

- Name of test material (as cited in study report): Lanthanum Acetate
- Physical state: Solid white powder
- Storage condition of test material: Room temperature in the dark under nitrogen

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Used whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability.

Metabolic activation:

with and without

Metabolic activation system:

male rat liver S9 mix

Test concentrations with justification for top dose:

EXPERIMENT 1
- 4-hour exposure without S9 followed by 20-hour culture in treatment-free media: 25, 50, 100, 200, 400 and 800 μg/mL.
- 4-hour exposure with S9 followed by 20-hour culture in treatment-free media: 25,50, 100, 200, 400 and 800 μg/mL.

EXPERIMENT II
- 24-hour without S9:25, 50, 100, 200, 400 and 800 μg/mL.
- 4-hour with S9 and followed by 20-hour culture in treatment-free media: 25, 50, 100, 200, 400 and 800 μg/mL.

The purity of the test item was 95.59% and was accounted for in the formulations.

Test concentrations were selected based on the results of a preliminary study. The selection of the maximum dose level was primarily based on precipitate rather than the onset of toxicity in all exposure groups tested because it had persisted on to the slides at the end of exposure and the toxicity could have been caused by the small decrease in pH.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Medium.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours for experiment 1, 24 hours and 4 hours for experiment 2


NUMBER OF CELLS EVALUATED: The first 100 consecutive well-spread metaphases from each culture were counted; where there were approximately 30 to 50% of cells with aberrations. Slide evaluation was terminated at 50 cells.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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: There was small significant change in pH when the test item was dosed into aqueous media, decreasing by more than 1 pH unit. Therefore, HEPES buffer was added (2% v/v) to buffer the pH change. The pH a was tested again in MEM during the Preliminary Toxicity test. Consequently, the change was small enough not to preclude testing the test item to the maximum dose level of 3160 μl/ml.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation: In Experiment I the maximum dose level selected for metaphase analysis was 200 μg/ml (with and without S9) due to the presence of precipitate on the slides. In Experiment II, the maximum dose level selected for metaphase analysis was therefore, 100 μg/ml in the absence and presence of S9, respectively.
- Other confounding effects: The test item was insoluble in dimethyl sulphoxide at 158 and 316 mg/ml and acetone at 316 mg/ml. However, the test item produced a doseable suspension at 31.60 mg/ml in an aqueous vehicle in solubility checks performed in-house. MEM at 31.60 mg/ml was, therefore, selected as the vehicle.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 12.34 to 3160 μg/ml. The maximum dose was based on the maximum recommended dose level, 10 mM concentration. Precipitate observations were made from the blood-free cultures. In the exposure groups in the absence of S9, precipitate was noted at the end of exposure at and above 24.69 μg/ml. However, in the 4(20)-hour exposure group in the presence of S9, precipitate was observed at all dose levels tested. In addition, precipitate persisted after exposure on the slides at and above 197.5 μg/ml in the absence of S9 and at and above 49.38 μg/ml in the presence of S9. Haemolysis was also observed in the blood cultures at and above 790 μg/ml in the 4(20)-hour exposure groups and at and above 395 μg/ml in the continuous exposure group. Haemolysis in this instance is thought to be the result of disruption of the membranes of erythrocytes and is not an indicator of toxicity to lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 3160 μg/ml in both of the 4(20)-hour exposure groups. However, they were considered to be not suitable for scoring in the case of the exposure group dosed without S9. In the continuous exposure group, metaphase cells were present up to 1580 μg/ml. There were no scorable metaphases present at the dose level above. Clear dose-related reductions in mitotic index were observed in the 24-hour continuous exposure group, whilst toxicity was observed in the 4(20)-hour exposure groups the dose-relationship was less clear.

The selection of the maximum dose level was primarily based on precipitate rather than the onset of toxicity in all exposure groups tested because it had persisted on to the slides at the end of exposure and the toxicity could have been caused by the small decrease in pH.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Vehicle: all vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes as determined by the in-house historical range of the laboratory.
- Positive controls: All the positive control items induced statistically significant increases in the frequency of cells with aberrations within the range determined by the in-house historical range of the laboratory, indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiments 1 and 2, the test item exhibited no dose-related inhibition of mitotic index
in the dose levels tested and, therefore was considered to be non-cytotoxic at the tested concentrations and under these experimental conditions.

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

The test item was considered not to induce any significant increases in the frequency of cells with aberrations, which included at least one precipitating dose level, either in the absence or presence of metabolic activation. Therefore, lanthanum acetate was considered to be non-clastogenic.