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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:
Between 29 June 1998 and 12 January 1999.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with OECD 473 with no deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
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:
- Physical state: amber viscous liquid
- Storage condition of test material: room temperature in the dark

Method

Target gene:
Chromosome damage.
Species / strain
Species / strain / cell type:
other: human whole blood
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The average generation time (AGT) for the regular donors used in this laboratory has been determined to be approximately 14 hours under optimal growth conditions.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared, in-house, from the livers of male Sprague-Dawley rats weighing - 200g. These had received a single ip injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation. The metabolic activation fraction (S9) was stored at -196 C.
Test concentrations with justification for top dose:
Experiment 1

with S9:
0, 20, 40, 80, 120, 160 and 240 ug/ml. 1 ml of 10% S9 (ie 1% final concentration of S9) in standard co-factors was added and the cultures
Positive control (Cyclophosphamide, CP), 25ug/ml

without S9:
0, 20, 40, 80, 120, 160 and 240 ug/ml
Positive control (Ethyl methanesulphonate, EMS), 750ug/ml

Experiment 2
with S9:
0, 5, 10, 20*, 40*, 80*, 120*, 160, 240 and 320 ug/ml. 1 ml of 20% S9 (ie 2% final concentration) in standard co-factors was added and the cultures
Positive control (CP), 25ug/ml

without S9:
0, 5, 10, 20*, 40*, 80*, 120*, 160, 240 and 320 ug/ml
Positive control (EMS), 500ug/ml


*: samples were analyzed for chromosome aberrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was a complex mixture, insoluble in water, and acetone was selected as the solvent because the test material was readily soluble in it at the required concentration.
Controls
Untreated negative controls:
no
Remarks:
Cyclophosphamide, Ethylmethanesulphonate
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Details on test system and experimental conditions:
METHOD OF APPLICATION: Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin-M
0.75 ml heparinised whole blood.

DURATION
- Exposure duration:
Cells cultured for 48 hours before exposure, then:
Experiment 1: 4 (16)h with and without S9
Experiment 2: 4(16)h with S9/ 20 h without S9

- Fixation time (start of exposure up to fixation or harvest of cells): . The cells were fixed by using fresh methano/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4 C for at least four hours to ensure complete fixation.
SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 ug/ml) two hours before the required harvest time

STAIN: for metaphase analysis, the slides were stained in 5% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and coverslipped using mounting medium
NUMBER OF REPLICATIONS: All treatments were performed in duplicate.

NUMBER OF CELLS EVALUATED: 200 per dose level

DETERMINATION OF CYTOTOXICITY
Method: 2000 nuclei counted to determine the mitotic index
Evaluation criteria:
The test substance will be regarded as clastogenic when it induced a statistically significant increase in the frequency of cells with aberrations either in the presence or absence of metabolic activation.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test or Chi-squared test.

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no observable change in pH when test material was dosed into media; no evidence of effect from pH.
- Effects of osmolality: no observable change in osmolality when test material was dosed into media; no evidence of effect from osmolality.
- Evaporation from medium: no data available
- Water solubility: no data available
- Precipitation: yes.
- Other confounding effects: none.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test material are given in the table below:

 

Final concentration(ug/ml)

20h-S9

0*, 20,40,80*,120*, 160*, 240, EMS 750*

20h+S9

0*, 20,40,80,120*,160*, 240*,CP25*

* dose levels selected for metaphase analysis

 

·       A cloudy precipitate of the test material was observed at 80 ug/ml and above at the washing stage of the exposure regimen. Haemolysis of the red cells was recorded at 240 ug/ml.

·       The qualitative assessment of the slides determined that there were scorable metaphases at all dose levels in both treatment groups.

·       The mitotic index data showed that approximately 50%mitotic inhibition was achieved at 160 ug/ml in the absence of S9 and at 240 ug/ml in the presence of S9.

·       The chromosome aberration data show that both of the vehicle control cultures had frequencies of cells with chromosome aberrations that were considered acceptable. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the presence or absence of metabolic activation.

·       The frequency of polyploid cells data show that the test material induced a significant increase in the numbers of polyploid cells at 120 ug/ml in the absence of metabolic activation, the middle dose assessed for aberrations.

 

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test material are given in the table below:

 

Final concentration(ug/ml)

20h-S9

0*, 5,10,20*,40*,80*,120*, 160, 240, EMS 500*

20h+S9

0*, 10, 20*,40*,80*,120*,160, 240, 320,CP25*

* dose levels selected for metaphase analysis

·       A cloudy precipitate was observed at and above 40 ug/ml without metabolic activation and 120 ug/ml with metabolic activation at the end of the exposure period for each treatment condition. Haemolysis was observed at and above 160 ug/ml.

·       The qualitative assessment of the slides determined that there were no scorable metaphases at and above 240 ug/ml and that there were very few metaphase cells at 160 ug/ml.

·       A dose related inhibition of mitotic index was observed and approximately 50% mitotic inhibition was achieved at 80 ug/ml in the absence and presence of S9.

·       The chromosome aberration data showed that all of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the presence or absence of metabolic activation.

The frequency of polyploid cell data showed that the test material did not induce a significant increase in the numbers of polyploid cells. However, a small increase over the control was observed in the continuous exposure group at 80 ug/ml, and in the presence of S9 at 40 ug/ml. There was no repeat of the response observed in the absence of metabolic activation in Experiment 1 and this may be due to the test material proving to be more toxic. The toxicological significance of polyploid responses is not clear, however, it is not a clastogenic effect.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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

Test Guidance

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1 997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 92/69/EEC.

Method and Material

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of S9 was repeated, whilst in the absence of activation the exposure time was increased to 20 hours.

Results

All vehicle (solvent) controls gave frequencies of cells with aberrations that were considered acceptable for human lymphocytes.

All the positive control treatments gave 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.

Conclusion

The test material did not induce any statistically significant increases in the frequency of cells with aberrations. Adequate levels of toxicity were achieved in all exposure groups. The test material was shown to be non-clastogenic to human lymphocytes in vitro.