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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-07-21 to 2014-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics
IUPAC Name:
Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics
Details on test material:
- Name of test material (as cited in study report): Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics
- Molecular weight (if other than submission substance): 240 g/mol
- Physical state: Liquid, colourless
- Analytical purity: 100 %
- Lot/batch No.: ZC4841
- Expiration date of the lot/batch: June 2015
- Stability under test conditions: Not indicated by the Sponsor
- Storage condition of test material: At room temperature, light protected

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Experiment II: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL

Without metabolic activation:
Experiment I: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Experiment II: 15.6, 27.3, 47.7, 83.6, 146.2, 255.9, 447.8, 783.7, 1371.4, 2400.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluatedAt least 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

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 applicable
Positive controls validity:
valid
Additional information on results:
The test item Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated due to strong clastogenic effects. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2400.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed. Phase separation was observed at the end of treatment in Experiment I and II in the presence of S9 mix at 83.6 µg/mL and above. In the absence of S9 mix phase separation was observed in Experiment I at 146.2 µg/mL and above and in Experiment II at 15.6 µg/mL and above.
No relevant influence on osmolarity or pH value was observed.
No relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration (Table 3 - Table 4).
Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, one single statistically significant increase was observed after treatment with 783.7 µg/mL (3.0 % aberrant cells, excluding gaps) (Table 7). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of S9 mix, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 µg/mL (Table 10). Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660.0 or 770.0 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Any other information on results incl. tables

Table2     Summary of results of the chromosomal aberration study with      
Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics    

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix

I

22 hrs

Solvent control1

100.0

2.5

2.5

0.5

 

 

Positive control2

120.8

10.0

10.0S

2.0

 

 

83.6

102.9

2.0

2.0

0.0

 

 

783.7PS

88.2

2.5

2.5

0.0

 

 

1371.4PS

91.8

3.0

3.0

0.0

 

 

2400.0PS

124.5

2.5

2.0

0.0

Exposure period 22 hrs without S9 mix

II

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

Positive control3#

31.9

40.0

39.0S

12.0

 

 

783.7PS

106.1

2.5

1.5

0.0

 

 

1371.4PS

105.2

1.0

1.0

0.0

 

 

2400.0PS

94.8

2.0

1.5

0.0

*   Including cells carrying exchanges

#    Evaluation of 50 metaphases per culture

##  Evaluation of 200 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone         0.5 % (v/v)

2     EMS          770.0 µg/mL

3     EMS          660.0 µg/mL


Table 2, cont.  Summary of results of the chromosomal aberration study with           
Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics         

Exp.

Preparationinterval

Test itemconcentration
in µg/mL

Mitotic indices
in %
of control

Aberrant cells
in %

 

 

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs with S9 mix

I

22 hrs

Solvent control1

100.0

0.5

0.5

0.0

 

 

Positive control2

31.6

9.5

9.5S

1.5

 

 

47.7

72.6

1.0

1.0

0.0

 

 

783.7PS

77.7

3.0

3.0S

0.0

 

 

1371.4PS

88.9

0.0

0.0

0.0

 

 

2400.0PS

95.2

1.5

1.5

0.0

II

22 hrs

Solvent control1

100.0

1.5

1.5

0.0

 

 

Positive control3

35.5

18.0

18.0S

3.0

 

 

27.3

107.6

1.0

0.5

0.0

 

 

47.7##

112.7

4.3

3.8

0.3

 

 

83.6PS

104.2

3.0

2.5

0.0

 

 

783.7PS

110.9

1.0

1.0

0.0

 

 

1371.4PS

116.4

1.5

1.5

0.0

 

 

2400.0PS

113.6

2.5

1.5

0.5

*   Including cells carrying exchanges

#    Evaluation of 50 metaphases per culture

##  Evaluation of 200 metaphases per culture

PS  Phase separation occurred at the end of treatment

S    Aberration frequency statistically significant higher than corresponding control values

1    Acetone         0.5 % (v/v)

2    CPA               2.5 µg/mL

3    CPA               7.5 µg/mL

Applicant's summary and conclusion

Conclusions:
A chromosome aberration study was performed for Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics according to OECD 473 and GLP. In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration (2400 μg/mL). Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of metabolic activation, one single statistically significant increase was observed after treatment with 783.7 μg/mL (3.0 % aberrant cells, excluding gaps). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of metabolic activation, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 μg/mL. Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.
Executive summary:

The test item Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics, dissolved in acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I & II

Exposure period

 4 hrs

22 hrs

 4 hrs

Recovery

18 hrs

-

18 hrs

Preparation interval

22 hrs

22 hrs

22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (2400.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data inaccordance with OECD Guideline 473. The rationale for the dose selection is reported in section3.5.1. The chosen treatment concentrations are reported inTable 1and the results are summarised inTable 2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

Either with or without metabolic activation no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix, one single statistically significant increase was observed after treatment with 783.7 µg/mL (3.0 % aberrant cells, excluding gaps). This value was clearly within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant. In Experiment II in the presence of S9 mix, one single increase (3.8 % aberrant cells, excluding gaps), slightly above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps), was observed after treatment with 47.7 µg/mL. Since this value was not statistically significant and no dose-dependency was observed, this finding has to be regarded as being biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.