Reaction mass of Fatty acids, montan-wax and Montan wax

Genetic toxicity: in vitro

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:

11 MAR 2009 to 28 MAY 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD guideline 473

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetz and Directive 2004/9/EEC

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Fatty acids, montan-wax and Montan wax

EC Number:

914-468-7

Molecular formula:

R-CH2-COOH mainly

IUPAC Name:

Reaction mass of Fatty acids, montan-wax and Montan wax

Details on test material:

- Name of test material (as cited in study report): Licowax S
- Physical state: particulate (stated "powder" in study report)

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 extract from Phenobarbital/b-Naphthoflavone induced Wistar rats

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml
Experiment II: 0.3, 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml

Without metabolic activation:
Experiment I: 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml
Experiment II: 0.3, 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- 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

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

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 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


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


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers

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 per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) and relative cell numbers were determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Licowax S, dissolved in THF, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster 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 metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours (Exp. I and II) after start of treatment with the test item.
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations.
The highest treatment concentration in this study, 35.0 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the solubility properties of the test item in an appropriate solvent (THF)
In both experiments visible precipitation of the test item in the culture medium was observed at 8.8 µg/mL and above in the absence of S9 mix and at 17.5 µg/mL and above in the presence of S9 mix. No relevant increase in the osmolarity or pH value was observed (Exp. I: solvent control: 391 mOsm, pH 7.4 versus 368 mOsm and pH 7.4 at 35.0 µg/mL; Exp. II: solvent control: 371 mOsm, pH 7.4 versus 360 mOsm and pH 7.4 at 35.0 µg/mL).
In this study at both preparation intervals in the absence as well as in the presence of S9 mix no statistically significant and biologically relevant cytotoxicity indicated by clearly reduced mitotic indices and/or cell numbers could be observed.
In both experiments in the absence and presence of S9 mix no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.8 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.5 ¿ 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory¿s historical solvent control data: 0.0 - 4.0 % aberrant cells excluding gaps).
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls.
In both experiments, either EMS (600 or 1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item Licowax S did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations.

Remarks on result:

other: strain/cell type: V79 cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Summary of results of the chromosomal aberration study with Licowax S

Exp.	Preparation		Test item	Cell numbers	Mitotic indices	Aberrant cells
	interval		concentration	in %	in %	in %
			in µg/mL	of control	of control	incl. gaps*	excl. gaps*	with exchanges
		Exposure period 4 hrs without S9 mix
I	18 hrs		Solvent control^1	100.0	100.0	4.0	2.5	0.0
			Positive control^2	n.t.	93.5	25.5	25.0^S	14.5
			2.2	95.8	105.7	2.5	1.0	0.5
			4.4	112.5	104.0	1.5	1.0	0.0
			8.8^P	92.1	103.2	0.5	0.5	0.0
		Exposure period 18 hrs without S9 mix
II	18 hrs		Solvent control^1	100.0	100.0	3.5	2.5	0.0
			Positive control^3	n.t.	121.6	20.0	19.5^S	8.5
			8.8^P	68.5	116.4	1.5	1.0	0.5
			17.5^P	88.8	105.2	1.5	1.5	0.0
			35.0^P	66.4	119.4	2.0	0.5	0.0
		Exposure period 4 hrs with S9 mix
I	18 hrs		Solvent control^1	100.0	100.0	2.0	1.5	0.0
			Positive control^4	n.t.	98.7	11.5	11.0^S	2.0
			4.4	86.6	93.5	2.5	2.0	0.5
			8.8	75.9	116.8	1.5	0.5	0.0
			17.5^P	94.3	112.6	3.5	3.5	1.5
II	18 hrs		Solvent control^1	100.0	100.0	3.5	3.0	1.0
			Positive control^4	n.t.	97.2	17.0	15.5^S	6.0
			8.8	115.2	92.4	3.0	3.0	0.5
			17.5^P	109.5	95.6	2.0	1.0	0.5
			35.0^P#	105.7	99.2	4.0	3.8	0.8

*     Inclusive cells carrying exchanges

^#     Evaluation of 200 metaphases per culture

n.t.  Not tested

^P      Precipitation occurred

^S     Aberration frequency statistically significant higher than corresponding control values

¹     THF         0.5 % (v/v)

²         EMS      1000.0 µg/mL

³     EMS600.0 µg/mL

⁴         CPA   1.4 µg/mL

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.
Therefore, Licowax S is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to precipitating concentrations.

Executive summary:

The test item Licowax S, dissolved in THF, was assessed for its potential toinduce structural chromosome aberrations inV79cells of the Chinese hamsterin vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I	Exp. II
Exposure period	4 hrs	18 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	14 hrs	14 hrs
Preparation interval	18 hrs	18 hrs	18 hrs	18 hrs

In each experimental group two parallel cultures were set up.At least100 metaphases per culture were evaluated for structural chromosome aberrations.

The highest applied concentration (35.0 µg/mL) was chosen withregard to the solubility properties of the test item in an appropriate solvent (THF) and with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations were:

with metabolic activation: Experiment I: 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml Experiment II: 0.3, 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml;

without metabolic activation: Experiment I: 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml Experiment II: 0.3, 0.5, 1.1, 2.2, 4.4, 8.8, 17.5, 35.0 µg/ml.

In the absence and the presence of S9 mix no cytotoxicity was observed up to the highest evaluated concentrations where test item precipitation occurred.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. 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 (p < 0.05) in cells with structural chromosome aberrations.