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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-12-15 to 2016-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
lutetium(3+) yttrium(3+) cerium oxidandiide silicate
EC Number:
941-731-3
Molecular formula:
Lu2(1-x)Y2xSiO5:Ce, 0
IUPAC Name:
lutetium(3+) yttrium(3+) cerium oxidandiide silicate
Test material form:
solid
Specific details on test material used for the study:
- Name: Lyso
- Chemical Name: Cerium doped Lutetium Yttrium Orthosilicate
- EC-No.: 941-731-3
- Batch-No.: C14-193
- Expiry Date: not applicable
- Physical State at Room Temperature: solid (crystal)
- Colour: colourless
- Purity: theoretical composition based on starting materials: 82.25% Lu2O3, 13.51% SiO2, 5.2% Y2O3 and 0.039% CeO2
- Molecular Weight: 440.7 g/mol
- Stability in Water at room temperature: stable
- Storage Conditions: at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was suspended in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Method

Target gene:
Histidine lo
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA 1537 and TA102
Details on mammalian cell type (if applicable):
CELLS USED:
- Source of cells: MOLTOX, INC., NC 28607, USA (TA98, 1535 and 102), Xenometrix AG, Switzerland (TA100 and 1537)
MEDIA USED
- Type and identity of media: Nutrient medium: 8 g Nutrient Broth and 5 g NaCl per litre, plus 125 µL ampicillin for TA98, TA100, TA102); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H20 and 12.2 mg biotin
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (please refer to box "Any other information on materials and methods"; Results: see box "Any other information on results", Table 2). 5000 µg/plate was selected as the maximum concentration. The concentration range covered with two logarithmic decades. The experiment was performed with the following concentrations:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (AppliChem; Lot No. 4I013997)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535, without S9, 10 µg/plate
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
TA98 (10 µg/plate) and TA1537 (40 µg/plate), without S9
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA102, without S9, 1 µL/plate
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
All strains, with S9, 2.5 µg/plate (10 µg/plate for TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment I)
- Cell density at seeding (if applicable): approx. 10^9 cells/mL, 100 µL/plate

EXPERIMENTAL PERFORMANCE
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain), 2000 μL Overlay agar.

DURATION
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level including the controls

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation). A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control
Evaluation criteria:
A test is considered acceptable if for each strain:
- The bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- The negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015) (see box “Any other information on material and methods”, Table 1)
- Corresponding background growth on negative control, solvent control and test plates is observed
- The positive controls show a distinct enhancement of revertant rates over the control plate
- At least five different concentrations of each tester strain are analysable.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed in any tester strain used. No toxic effects of the test item were noted in any of the five tester strains used upt o the highest dose group evaluated. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed. The reference mutagens induced a distinct increase of revertants indicating the validity of the experiments. The negative controls were within the histrical control data range.

Any other information on results incl. tables

Results of the pre-experiment:

Table 2: Results of the pre-experiment

Dose (µg/plate) TA 98 TA 100
Mutation Factor [toxicity]*  Mutation Factor [toxicity]*
without S9 with S9 without S9 with S9
Solvent Control (DMSO) 1.0 1.0 1.0 1.0
4-NOPD 10 9.6 - - -
NaN3 10 - - 6 -
2-AA 2.5 - 57.7 - 11.9

Test Item

Cerium doped Lutetium Yttrium Orthosilicate


3.16 1.3 1.1 1.1 1.1
10.0 1 1.1 1.1 1
31.6 1.5 0.9 1.1 1.2
100 1.4 1.1 1.0 1
316 1.3 1.1 1.0 1.1
1000 1.2 1.1 1.0 1.2
2500 1.2 1.2 1.2 0.9
5000 1.2 1.2 1.1 1.2
* [toxicity parameter]: B = Background lawn reduced, N = No background lawn, P = Precipitation, P* = not evaluable, precipitation interferes with scoring

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, Cerium doped Lutetium Yttrium Orthosilicate did not cause gene mutations in an Ames Test conducted according to OECD 471. Therefore, Cerium doped Lutetium Yttrium Orthosilicate is considered to be non-mutagenic according to CLP criteria in this bacterial reverse gene mutation assay .
Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA98, TA100, TA102, TA1535 and TA1537 of Salmonella typhimurium were exposed to Cerium doped Lutetium Yttrium Orthosilicate in DMSO at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains. Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.