[No public or meaningful name is available]

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:

experimental study

Adequacy of study:

key study

Study period:

From 02 January to 10 April 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD test guideline No. 473 and in compliance with GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 2007-08-21)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): ST 22 C 07
- Substance type: pure active substance
- Physical state: clear colourless liquid
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: humans

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin Band 15% foetal calf serum, at 37°C with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 µg/mL final concentration.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from rat Sprague-Dawley (Exp. 1: 2%, Exp.2 : 1%)

Test concentrations with justification for top dose:

Prelim. : 7.12 to 1823 µg/mL (up to 10 mM).
Exp. 1: Without S9 : 0*, 30, 60*, 90*, 120*, 150*, 180 µg/mL.
Exp. 1: With S9 : 0*, 30, 60, 90*, 120*, 150*, 180* µg/mL.
Exp. 2: Without S9 : 0*, 30, 60, 90*, 120*, 150*, 180 µg/mL.
Exp. 2: With S9 : 0*, 60, 90*, 120*, 150*, 180, 210 µg/mL.
* Dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: well known solvent/vehicle used as test substance not soluble in water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9, 5 µg/mL in DMSO

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9, 0.4 µg/mL in Exp.1 and 0.2 µg/mL in Exp. 2 in MEM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: mytogenic stimulation of lymphocytes (with 90 µg/mL of phytohaemagglutinin (PHA)) was performed 48 hours before lymphocyte treatment with the test substance.
- Exposure duration: with metabolic activation: 4 hours; without metabolic activation: 4 hours or 24 hours continuous
- Expression time (cells in growth medium): with metabolic activation: 20 hours ; without metabolic activation: 20 h in Exp. 1, NA for Exp. 2.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid) 0.1 µg/mL for 2 hours (2 hours before cell harvest)
STAIN (for cytogenetic assays): Gurrs Giemsa (5 % for 5 min)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture, i.e. 200 cells/dose (metaphase analysis)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: YES
- Determination of endoreplication: YES
- Other: any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

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. Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

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: humans

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see Attached background material

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 & effects of osmolality: There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm. No relevant impact on the study.
- Evaporation from medium: Vapour pressure of test substance measured according OECD 104 is of 35 Pa. No relevant impact on the study.
- Water solubility: 119 mg/L (OECD 105)
- Precipitation: not seen in Exp. 1 or 2.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed to determine maximal dose levels in Experiments. The dose range for the preliminary toxicity test was 7.12 to 1823 µg/mL. The maximum dose was the maximum recommended dose level equivalent to a 10 mM concentration. A precipitate of the test material was observed in parralel blood free cultures at the end of the exposure, at and above 455.75 µg/mL in all three exposure group (see attached background material). Haemolysis was also observed in the blood cultures at and above 56.97 µg/mL in both the 4(20)-hour exposure groups and at and above 113.94 µg/mL in the continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 113.94 µg/mL in all three exposure groups. The mitotic index data are presented in Table 1. The test material induced clear evidence of toxicity in all three of the exposure groups.
The selection of the maximum dose level was based on toxicity and was 180 µg/mL for the 4(20) hour exposure groups in Experiment 1 and for the for the 24-hour continuous exposure group used in Experiment 2. A higher dose level of 210 µg/mL was used in Experiment 2 for the 4(20)-hour exposure group in the presence of S9

COMPARISON WITH HISTORICAL CONTROL DATA: YES. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The positive control in the absence of S9 only generated a modest increase in the frequency of cells with aberrations. However, the response was within the historical positive control range, clearly statistically significant and, therefore, considered to be acceptable. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Exp. 1: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 180 µg/mL in the presence of metabolic actication (+ S9). In the absence of metabolic activation (- S9) the maximum dose level of the test material with metaphases suitable for scoring was 150 µg/mL. Haematolysis was noted in the blood cultures at the end of exposure and at above 60 µg/mL for the 4(20)-hour exposure group in the absence of S9 and at and above 90 µg/mL for the 4(20)-hour in the presence of S9. 54% mitotic inhibition was achieved at 150 µg/mL in the absence of S9. In the presence of S9 43% mitotic inhibition was achieved at 180 µg/mL.
Exp. 2: The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to 150 µg/mL in the presence and absence of metabolic activation. Haematolysis was not seen at the beginning or end of exposure in the cultures with the dose levels used in Exp. 2. 48% and 31% mitotic inhibition was achieved at 150 µg/mL in the absence and presence of S9 respectively. The toxicity observed in the 4(10)-hour exposure in the presence of S9 was greater in Exp.2 than that observed in Exp. 1 with no metaphase being present at 180 µg/mL. This was considered to be a result of the reduced S9 concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

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

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, duplicate culture of human lymphocyte were treated with the test material diluted in DMSO at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included some dose levels that induced approximately 50 % mitotic inhibition.

Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and of the Directive 67/548/EEC.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.