Hydrocarbons, C15-C19, n-alkanes, isoalkanes,

Genetic toxicity: in vitro

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 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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 4

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

Test material information

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

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 control^1	100.0	2.5	2.5	0.5
		Positive control^2	120.8	10.0	10.0^S	2.0
		83.6	102.9	2.0	2.0	0.0
		783.7^PS	88.2	2.5	2.5	0.0
		1371.4^PS	91.8	3.0	3.0	0.0
		2400.0^PS	124.5	2.5	2.0	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
		Positive control^3#	31.9	40.0	39.0^S	12.0
		783.7^PS	106.1	2.5	1.5	0.0
		1371.4^PS	105.2	1.0	1.0	0.0
		2400.0^PS	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 control^1	100.0	0.5	0.5	0.0
		Positive control^2	31.6	9.5	9.5^S	1.5
		47.7	72.6	1.0	1.0	0.0
		783.7^PS	77.7	3.0	3.0^S	0.0
		1371.4^PS	88.9	0.0	0.0	0.0
		2400.0^PS	95.2	1.5	1.5	0.0
II	22 hrs	Solvent control^1	100.0	1.5	1.5	0.0
		Positive control^3	35.5	18.0	18.0^S	3.0
		27.3	107.6	1.0	0.5	0.0
		47.7^##	112.7	4.3	3.8	0.3
		83.6^PS	104.2	3.0	2.5	0.0
		783.7^PS	110.9	1.0	1.0	0.0
		1371.4^PS	116.4	1.5	1.5	0.0
		2400.0^PS	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.