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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 6, 2011 - Dec 2, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): XP-7866
- Analytical purity: 99.5
- Purity test date: 16.05.2011
- Lot/batch No.: 10188-1 JM
- Expiration date of the lot/batch: 28.02.2015

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Primary lymphocytes from suitable donors. The cell cycle time was determined by BrdU (bromodeoxyuridine) incorporation to assess the average generation time (AGT) and the number of first, second and third division metaphase cells.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone and ß-Naphthoflavone induced rat liver S9-preparation from male Sprague-Dawley rats.
Test concentrations with justification for top dose:
The test concentrations were selscted following a preliminary cytotoxicity test.
Experiment 1 and 2 without and with S9: 0, 17.92, 35.84, 71.69, 143.38, 286.75, 573.5, 1147, 2294 micro-g/ml
Vehicle / solvent:
Dimethylsulfoxide (DMSO) 99.9%. The solvent was chosen as the test item produced a suspension suitable for dosing in this vehicle.
Controls
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: mitomycin C, with S9 Cyclophosphamide
Details on test system and experimental conditions:
CELL CULTURE: cells were grown in Eagles minimal essential medium with HEPES buffer supplemented with glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum at 37 deg. C with 5% CO2 humidified air. the lymphocytes of fresh heparinised blood were stimulated to divide by addtion of phythaemagglutinin (PHA).
DURATION
- Exposure duration:
without S9: Experiment 1: 4 h, Experiment 2: 24 h
with S9: Experiment 1 and 2: 4 h
- Expression time (cells in growth medium): 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): With metabolic activation: 24 h, without metabolic activation Experiment 1 24 h, experiment 2 44 h

SPINDLE INHIBITOR (cytogenetic assays): Demeclocine (Colcemid 0.1 micro-g/ml) applied 2 h before harvest time
STAIN (for cytogenetic assays): Giemsa (5%)

NUMBER OF REPLICATIONS: duplicate cultures per concentration

NUMBER OF CELLS EVALUATED: 100 well spread metaphases from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index from 2000 lymphocylte cell nuclei.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes cells with 69 chromosomes otr more.
.
Evaluation criteria:
A positive response is reported for a particular treatment if the percentage of cells with aberrations other than gaps markedly exceeded that seen in concurrent controls with or without a clear dose-response relationship. For modest increases in aberration frequency a dose response relationship is generally required and statistical tests may be applied.
Statistics:
Fishers exact test was used to compare frequencies of cells with aberrations excluding gaps and frequency of polyploid cells with concurrent vehicle controls.

Results and discussion

Test results
Species / strain:
lymphocytes: human, primary
Metabolic activation:
with and without
Genotoxicity:
not specified
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
- Precipitation: Aprecipitation of the test article was observed in the preliminary test at the end of the exposure period at a concentration of 35.84 micro-g/ml and above. In the main study precipitation was observed at the end of the treatment period at and above 143.38 micro-g/ml and above 17.92 micro-g/ml in cell pellets durign the washing procedure.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary study the test item showed evidence of cytotoxicity in all 3 exposure groups, but no clear dose response was observed. Countable metaphases were present up to 2294 micro-g/ml. This concentration was also the maximum obtainable concentration in the vehicle.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Up to the maximum practiclly obtainable concentration of 2294 micro-g/l 50% inhibition of growth was not achieved. In experiment 1 a maximum of 16% inhibition was achieved in the absence of S9 at 573.5 micro-g/ml and 25% inhibition at 34.84 micro-g/ml in the presence of S9.

All of the vehicle control cultures had frequencies of cells with chromosomal aberrations in the expected range. The positive control items incuded statistically significant increases in the frequency of cells with aberrations.

In both replicate experiments the test item did not induce any statistically signinficant increases in frequency of cells with aberrations, either in presece or absence of metabolic activation. The test item did not induce statistically significant increases in the numbe rof polyploid cells at any dose level in any of the exposure groups.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this test the test item did not induce any significant increase in chromosomal aberrations or polyploidie, both in the presence and absence of a metabolic activation system and was therefore not clastogenic in human lymphocytes in vitro.
Executive summary:

The test item was for its ability to induce chromosomal aberrations in vitro in cultured mammalian cells. The study was performed according to OECD TG No. 473 (1997) and EU Method B.10 (2008) and under GLP conditions. Duplicate cultures of primary human lymphocytes treated with the test item at 4 concentration levels and including positive and negative contols were evaluated for the induction of chromosomal aberrations with and without a metabolic activation system. In experiment 1 a 4 h exposure period followed by a 20 h expression period both with and without metabolic activation was performed, while in experiment 2 the exposure time without metabolic activation was prolonged to 24 hours. Cytotxicity was assessed by determining the mitotic index of 2000 lymphocyte nuclei. For the assessment of chromosomal aberrations 100 metaphases from each culture (200 per concentration) were counted. The negative and positive controls gave results in the expected ranges. The test item did not induce any significant increase in chromosomal aberrations or polyploidie in any of the experiments at any dose level with and without metabolic activation. Therfore the test item is considered to be non-clastogenic to human lymphocytes.