Paraffin waxes and Hydrocarbon waxes, oxidized, lithium salts

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:

8th September 2003 to 25th November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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:

other: UK department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date signed: 13/02/03 Alexander, R.G.

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbon waxes (petroleum), oxidized

EC Number:

265-205-1

EC Name:

Hydrocarbon waxes (petroleum), oxidized

Cas Number:

64743-00-6

Molecular formula:

too complex

IUPAC Name:

Hydrocarbon waxes (petroleum), oxidized

Test material form:

other: greasy solid

Details on test material:

- Physical appearance: Brown waxy solid.
- Storage condition of test material: Room temperature in the dark.

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

- Average generation time: ~ 17 hours for this donor, determined under typical experimental conditions.
- Medium: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum, at 37ºC with 5% CO2 in air.
- Cells were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 µg/ml final concentration.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1
4 hour exposure with and without S9 mix: 0, 156.25, 312.5, 625, 1250, 1875, 2500 µg/ml

Experiment 2
4 hour exposure with S9 mix: 0, 39.06, 78.13, 156.25, 312.5, 625 and 1250 µg/ml
24 hour exposure without S9 mix: 0, 39.06, 78.13, 156.25, 312.5, 468.75 and 625 µg/ml

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

PRELIMINARY STUDY:
A dose range finding study was performed using concentrations of the test material between 19.5 and 5000 µg/ml.

MAIN TEST: The study was carried out in two experiments, utilising four different exposure conditions.

Experiment 1
- The cells were exposed for 4 hours without S9-mix followed by 20 hour culture in media free from the test material, prior to cell harvest.
- The cells were exposed for 4 hours with S9-mix followed by 20 hour culture in media free from the test material, prior to cell harvest.

Experiment 2
- Continuous exposure to the test material for 24 hours prior to cell harvesting.
- The cells were exposed to the test material for 4 hours with S9-mix followed by 20 hour culture in test material free media, prior to cell harvest.

TEST CONDITIONS:
- Preincubation period: 48 hours at 37 ºC, 5% CO2 in humidified air.
- Volume of test material, vehicle or positive control: 0.1 ml
- Total nominal volume: 10 ml.
- Volume and concentration of S9 mix: 1 ml of 20% S9-mix in experiment 1 and 1 ml of 10% S9-mix in experiment 2.
- Expression time (cells in growth medium): 4 hours with the test material and 20 hours without the test material.
- In the case of the continuous exposure the expression time was 24 hours, with the test material.

SPINDLE INHIBITOR (cytogenetic assays): demecolcine, Colcemid 0.1 µg/ml.
STAIN (for cytogenetic assays): 5% Gurrs Giemsa.

NUMBER OF REPLICATIONS: Tests performed in duplicate.

NUMBER OF CELLS EVALUATED: 2000 lymphocytes cell nuclei were counted and the number of cells in methaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Evaluation criteria:

Scoring:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976).

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test. Samples which showed a statistical increase in aberrations were considered to have given a positive result.

Statistics:

Fisher’s Exact test.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1:

-The test material did not induce a statistically significant increase in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

- The maximum dose level with scoreable metaphases present was 1250µg/ml in the presence and absence of metabolic activation. - The mitotic data confirms the qualitative assessment that the dose-related inhibition of mitotic index was observed and that 33% mitotic inhibition was achieved at 1250 µg/ml in the absence of S9 and 35% in the presence.

- Complete inhibition was observed at 1875 and 2500µg/mlin both exposure groups.

Experiment 2:

-The test material did not induce a statistically significant increase in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

- Qualitative assessment of the slides determined that the maximum dose level with scorable metaphases was 1250µg/ml with S9 mix and 468.75µg/ml without S9 mix in experiment 2.

- The mitotic index data confirms the qualitative observations that a dose-related inhibition of mitotic index was observed and that 67% mitotic inhibition as achieved at 468.75 µg/ml in the absence of S9. In the presence of S9 50% mitotic inhibition was achieved at 1250 µg/ml.

Table 1. Results of Chromosome Aberrations

Experiment No.	With or Without S9 mix	Treatment Group	Dose Level (µg/ml)	Total No. of Aberrations		Frequency of Aberrant Cells (%)
				(+ Gaps)	(- Gaps)	(+ Gaps)	(- Gaps)
1	Without	Vehicle Control		2	1	1	1
		Test Material	312.5	1	1	1	1
			625	3	2	3	2
			1250	2	1	2	1
			1875	TOXIC
		Positive Control MMC	0.4	39	29	28	22***
1	With	Vehicle Control		7	5	3	1
		Test Material	312.5	2	2	2	2
			625	1	0	1	0
			1250	5	3	5	3
			1875	TOXIC
		Positive Control CP	10	89	50	65	39***
2	Without	Vehicle Control		3	3	3	3
		Test Material	156.25	1	0	1	0
			312.5	1	0	1	0
			468.75	1	1	1	1
			625	TOXIC
		Positive Control MMC	0.2	72	53	50	39***
2	With	Vehicle Control		2	1	2	1
		Test Material	312.5	1	0	1	0
			625	2	2	2	2
			1250	8	3	8	3
		Positive Control CP	0.2	96	73	52	42***

MMC = Mitomycin C

CP = Cyclophosphamide

*** = P < 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the test, the test material was determined to be non-clastogenic to human lymphocytes. There was no statistically significant increase in the frequency of cell aberrations in the absence or presence of metabolic activation.

Executive summary:

In a GLP compliant chromosome aberration study performed according to the standardised guidelines; OECD 473, EU Method B.10 and UK department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals, the test material was determined to be non-clastogenic. Under the conditions of the test the human lymphocyte did not produce a statistically significant increase in the frequency of cell aberrations in the absence or presence of metabolic activation. Therefore the test material is considered to be non-mutagenic.