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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:
9 November 1994- 21 February 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Not relevant
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 tissue culture medium
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by Aroclor 1254
Test concentrations with justification for top dose:
First test:
Without S-9 mix: 3.75, 7.5, 15, 20, 30, 40, 50, 60, 80 and 100 ug/ml
With S-9 mix: 7.8, 15.6, 31.25, 62.5, 75, 100, 125, 150, 200 and 250 ug/ml
Second test:
Without S-9 mix, 18 hour harvest: 3.75, 7.5, 15, 20, 30, 40, 50, 60, 80 and 100 ug/ml
With S-9 mix, 18 hour harvest: 15.6, 31.25, 62.5, 75, 100, 125, 150, 200 and 250 ug/ml
Without S-9 mix, 32 hour harvest: 7.5, 15, 20, 30, 40, 50, 60 and 80 ug/ml
With S-9 mix, 32 hour harvest: 62.5, 75, 100, 125, 150, 200 and 250 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulphonate (direct-acting mutagen) and Cyclophosphamide (promutagen)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Test 1: with S-9: 3 hours; without S-9: continuous exposure: 18 hours
Test 2: with S-9: 3 hours; without S-9: 18 or 32 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
Test 1: 18 hours
Test 2: 18 or 32 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine, 0.25 μg/ml., last 2 hours of incubation
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: duplicate cultures for each treatment

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads from each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value was used as the highest dose level for the metaphase analysis.

OTHER: Prior to commencing testing, the solubility of the test substance was assessed. OTNE was found to be miscible with DMSO at 125 ug/ml. When added to culture medium at 1% v/v, to give a final concentration of 1250 ug/ml, small oily globules were formed. The highest concentration chosen for subsequent testing, 1000 ug/ml, was at the limit of solubility since only a slight amount of insoluble test substance was present.
Evaluation criteria:
No data
Statistics:
Fisher's test. (FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York).

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
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:
not examined
Positive controls validity:
valid
Additional information on results:
Test 1:
In the absence of S-9 mix, OTNE caused a decrease in the mitotic index (MI) to 27% of the solvent control value at 62.5 ug/ml. However, there were insufficient metaphase figures available for analysis. As no other concentration caused a suitable reduction in the MI, the test was repeated using a narrower range of dose levels. In the repeat test, 50 ug/ml caused a reduction in the MI to 55% of the solvent control value. This concentration, together with 30 and 15 ug/ml, was selected for further analysis.
In the presence of S-9 mix, OTNE caused a decrease in MI to 67% of the solvent control value at 125 ug/ml. However, there were insufficient metaphase figures suitable for analysis. As no other concentration caused a suitable reduction in the MI, the test was repeated with a narrower range of dose levels. In the repeat test 125 ug/ml caused a reduction in the MI to 40% of the solvent control value. This concentration, together with 62.5 and 15.6 ug/ml was selected for further analysis.
Test 2: 18 hour harvest
In the absence of S-9 mix, OTNE caused a dose-related decrease in MI. The highest concentration with sufficient metaphase figures suitable for analysis, 30 ug/ml, caused a reduction in the MI to 58 % of the solvent control value. Therefore, the dose levels chosen for metaphase analysis were 30, 15 and 7.5 ug/ml.
In the presence of S-9 mix, OTNE, at a concentration of 125 ug/ml, caused a reduction in the MI to 61 % of the solvent control value. All higher dose levels were too toxic for analysis. Therefore, the dose levels chosen for metaphase analysis were 125, 62.5 and 31.25 ug/ml.
Test 2 - 32 hour harvest
In the absence of S-9 mix, although the MI was reduced to 49% of control levels at 30 ug/ml, there were insufficient metaphase figures suitable for analysis. The highest analysable concentration, 15 ug/ml, reduced the MI to 73% of the solvent control value. Therefore, 15 ug/ml was chosen for metaphase analysis.

COMPARISON WITH HISTORICAL CONTROL DATA: Any increase in the number of aberrant cells lies within the historical control range.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Except for one test, there were no statistically significant increases in the proportion of metaphase figures with chromosomal aberrations, at any concentration analysed, in the presence and absence of S-9 mix. In the second test -18 hour harvest - in the absence of S-9 mix, there was a statistically significant increase in the number of aberrant cells at the highest concentration, 30 ug/ml, when gap damage was excluded. However, this increase, to 4.0%, lies within the historical control range of 0-5.25 %, there was no dose-response relationship and this increase was not seen in the first test or at the later sampling time. Therefore, it is concluded that this increase is not treatment-related.

Applicant's summary and conclusion

Conclusions:
There were no statistically significant increases, treatment-related, in the proportion of aberrant cells, in the presence and absence of S-9 mix, at the 18 or 32 hour harvests. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. It is concluded that OTNE has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
Executive summary:

A study was performed to assess the ability of OTNE to induce chromosomal aberrations in human lymphocytes cultured in vitro. Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treatment time, cell division was arrested using Colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage.

In order to assess the toxicity of OTNE to cultured human lymphocytes, the mitotic index (MI) was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test:

Without S-9 mix, 18 hour harvest: 15, 30 and 50 ug/ml

With S-9 mix, 18 hour harvest: 15.6, 62.5 and 125 ug/ml

Second test:

Without S-9 mix, 18 hour harvest: 7.5, 15 and 30 ug/ml

With S-9 mix, 18 hour harvest: 31.25, 62.5 and 125 ug/ml

Without S-9 mix, 32 hour harvest: 15 ug/ml

With S-9 mix, 32 hour harvest: 75 ug/ml

There were no statistically significant increases in the proportion of aberrant cells, in the presence of S-9 mix, at the 18 or 32 hour harvests. In the absence of S-9 mix, in the second test at the 18 hour harvest, there was a statistically significant increase in the number of aberrant cells at the highest concentration, 30 ug/ml, when gap damage was excluded. However, this increase lies within the historical control range, there was no dose-relationship and this increase was not seen in the first test or at the later sampling time. Therefore, it is concluded that this increase is not treatment-related.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells.

It is concluded that OTNE has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.