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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:
28 August to 28 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate OECD and EC Test Guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.
Deviations:
no
GLP compliance:
yes
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): Tetrabutane
- Substance type: technical material
- Physical state: liquid
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: C16-(branched), C20-(branched) and C24-(branched)-alkanes
- Isomers composition: no data
- Lot/sample No.: lot No. 16.06.2008/sample No. 0649/82535
- Expiration date of the lot/batch: 8 June 2010
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes:
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver fraction (S9) from male CD rats
Test concentrations with justification for top dose:
First test
In the absence of S9 mix: 27.86, 46.44, 77.40, 129, 215, 358.34, 597.24, 995.4, 1659 and 2765 μg/mL.
In the presence of S9 mix: 27.86, 46.44, 77.40, 129, 215, 358.34, 597.24, 995.4, 1659 and 2765 μg/mL.

Second test
In the absence of S9 mix: 250, 500, 1000, 1500, 2000, 2500 and 2765 μg/mL.
In the presence of S9 mix: 250, 500, 1000, 1500, 2000, 2500 and 2765 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:
Prior to commencing testing, the solubility of the test substance in solvents compatible with
the test system was assessed. Tetrabutane was found to be miscible in acetone at
276.5 mg/mL (1M) following sonication and incubation at 37°C. On dosing a 276.5 mg/mL
solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of
2765 μg/mL, no precipitate was observed.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations
(Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not
used in this test system.
The osmolality and pH of the test substance in medium was tested at 2765 μg/mL; no
fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0
unit was observed compared with the solvent control.
In this case, the highest final concentration used for subsequent testing was 2765 μg/mL
(10 mM). In this study Tetrabutane was added to cultures at 1% v/v (50 μL per 5 mL
culture).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
mitomycin C (without S9), Cyclophosphamide (with S9) Migrated to IUCLID6: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: 48 hours
- Exposure duration:
The study comprised two independent tests. In the first test, a three hour treatment was used
in both the absence and presence of S9 mix. In the second test, a 21 hour continuous
treatment was used in the absence of S9 mix, and a three hour treatment using an increased
S9 concentration (5% v/v) was used in the presence of S9 mix.

- Fixation time (start of exposure up to fixation or harvest of cells):
First test: three hours after dosing, the cultures were centrifuged at 500g for 5 minutes. The
supernatant removed and the cell pellets resuspended in fresh medium. They were then
incubated for a further 18 hours.
Second test: three hours after dosing, the cultures containing S9 mix were centrifuged. The cell pellets
were rinsed and resuspended in fresh medium. They were then incubated for a further
18 hours. Cultures treated in the absence of S9 mix were incubated continuously for
21 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: duplicate


NUMBER OF CELLS EVALUATED: 100 each replicate


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index:
The prepared slides were examined by light microscopy using a low power objective. The
proportion of mitotic cells per 1000 cells in each culture was recorded except for positive
control treated cultures, or cultures where there were no signs of cytotoxicity.

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploid and endoreduplicated cells were noted when seen.



OTHER:
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within
the current historical control range.

The test substance is considered to cause a positive response if the following conditions are
met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant
chromosomes (excluding gaps) are observed at one or more test concentration.

The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.

The increases are reproducible between replicate cultures.

The increases are not associated with large changes in pH, osmolality of the treatment
medium or extreme toxicity.

Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of
aberrant cells above concurrent control frequencies are observed, at any concentration.

A further evaluation may be carried out if the above criteria for a positive or a negative
response are not met.
Statistics:
The number of aberrant metaphase cells in each treatment group was compared with the
solvent control value using the one-tailed Fisher exact test (Fisher 1973).

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test
substance groups. If this is significant at the 1% level, the test is reiterated excluding the
highest dose group - this process continues until the trend test was no longer significant.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Human lymphocytes
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 applicable
Positive controls validity:
valid

Any other information on results incl. tables

First test

Toxicity data

       

Mitotic indices of cultured human lymphocytes treated with Tetrabutane are shown in Tables 2a and 2b (attachment below).

In the absence of S9 mix following three hour treatment, Tetrabutane caused no significant reduction in the mitotic index at 2765mg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 995.4, 1659 and 2765 mg/mL.

In the presence of S9 mix (2% v/v final concentration) following three hour treatment,Tetrabutane caused no reduction in the mitotic index at 2765mg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 995.4, 1659 and 2765 mg/mL.

Metaphase analysis

     

The effects of Tetrabutane on the chromosomes of cultured human lymphocytes are summarised in Table1 (attachment below).

In the absence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at either 995.4 or 1659mg/mL, when compared with the solvent control. The mean values for the solvent control (acetone) and both Tetrabutane treatment concentration were within the historical control range for this laboratory. However, at 2765mg/mL, a statistical significance (p<0.001: including gaps and p<0.01: excluding gaps) which exceeded the historical solvent control range for this laboratory was observed. As the data suggested no clear concentration-related response, the result was considered biologically non-relevant.

In the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control. All mean values for the solvent control (acetone), and all Tetrabutane treatment concentrations were within the historical control range for this laboratory.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Second test

Toxicity test

        

Mitotic indices of cultured human lymphocytes treated with Tetrabutane are shown in Tables 4a and 4b (attachment below).


In the absence of S9 mix following 21 hour continuous treatment, Tetrabutane caused no reduction in the mitotic index at 2765mg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 2000, 2500 and 2765mg/mL.

In the presence of S9 mix (5% v/v final concentration) following three hour treatment, Tetrabutane caused no reduction in the mitotic index 2765 atmg/mL, compared to the solvent control value. The concentrations selected for the metaphase analysis were 2000, 2500 and 2765mg/mL.

Metaphase analysis

       

The effects of Tetrabutane on the chromosomes of cultured human lymphocytes are summarised in Table1 (attachment below).

In both the absence and the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.

All mean values for the vehicle control (acetone), were within the historical control range for this laboratory. All mean values for Tetrabutane were within the historical control range for this laboratory, with the exception of 2500mg/mL (excluding gaps) and 2765mg/mL (including and excluding gaps) in the absence of S9 mix only, were values were marginally outside the historical control range for this laboratory.

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploid anaylysis

No statistically significant increases in polyploid metaphases were observed during metaphase analysis in either test.

Applicant's summary and conclusion

Conclusions:
It is concluded that the test substance Tetrabutane has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

A study was performed at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH,, to investigate the possible clastogenic properties of the test substance Tetrabutane. The study investigated the ability of the test substance (initially at 10 concentrations up to a maximum of 2765 µg/mL) to induce chromosomal aberrations on cultured human lymphocytes from healthy donors in two independent experiments. The investigations were performed with and without rat liver S9 fraction as metabolizing system. The study was conducted to GLP and according to OECD guidelines 473 and EU method B.10. At the highest practical concentration of 2765 µg/mL there was no significant reduction of the mitotic index compared to the vehicle control, in the presence or absence of metabolic activation. The concentrations selected for metaphase analysis in the first test were 995.4, 1659 and 2765 µg/mL and in the second test 2000, 2500 and 2765 µg/mL.

In the first test at 2765 ug/mL, in the absence of S9 mix, a statistical significance (p<0.001: including gaps and p<0.01: excluding gaps) which exceeded the historical solvent control range for this laboratory was observed. As the data suggested no clear concentration-related response, the result was considered biologically non-relevant. Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at either 995.4 or 1659 ug/mL. In the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control.

 

In the second test, both in the absence and the presence of S9 mix, Tetrabutane caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.

 

It is concluded that the test substance Tetrabutane has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.