Hydrocarbon waxes (petroleum), oxidized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th April 2012 to 2nd July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese METI/MHLW guidelines for testing of new chemical substances

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom.

Type of assay:

mammalian cell gene mutation assay

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 solid block.
- Storage Conditions: Room temperature in the dark.

Method

Target gene:

Thymidine kinase, TK +/-, locus in L5178Y mouse lymphoma cells. Mutant form is TK-/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

-Master stocks of cells were tested and found to be free of mycoplasma.
-Cell Cleansing: TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Cells were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/ml), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/ml) and Glycine (22.5 µg/ml).

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the livers of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
4 hour exposure without S9 mix: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 234.38, 312.5, 468.75 µg/ml.
4 hour exposure with S9 mix: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 468.75, 625 µg/ml.

Experiment 2
24 hour exposure without S9 mix: 0, 6.25, 12.5, 25, 50, 100, 150, 200, 250, 300, 350 µg/ml.
4 hour exposure with S9 mix: 0, 50, 100, 200, 300, 350, 400, 450, 500 µg/ml.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

CELL CULTURE
-The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air.
-The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

PREPARATION OF TEST AND CONTROL ITEMS
-The test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. Due to formulation difficulties, the maximum dose level suitable for dosing was set at 2500 µg/ml.
-Acetone is toxic to L5178Y at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 250 mg/ml and dosed at 0.5% to give a maximum achievable dose level of 1250 µg/ml.
-The test item was a complex mixture and, therefore, purity was not accounted for when formulating the dosing solutions.

MICROSOMAL ENZYME FRACTIONS
-PB/βNF S9 was prepared in-house from the livers of male Sprague-Dawley rats weighing approximately 250 g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. The S9 was stored at approximately -196°C in a liquid nitrogen freezer.
-S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
-20% S9 mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. In Experiment 2, 10% S9 mix (i.e. 1% final concentration of S9), was added.

PRELIMINARY TOXICITY TEST
-A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 4.88 to 1250 µg/ml for all three of the exposure groups.
-Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/ml.
-The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.
-Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

MUTAGENICITY TEST
Experiment 1
-An exponentially growing stock culture of cells was set up. Cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9 mix). To each universal was added 2 ml of S9 mix if required, 0.1 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml.
-The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
-As in experiment 1, cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10^6 cells/ml in 10 ml cultures were established in 25 cm2 tissue culture flasks.
-The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix). To each universal was added 2 ml of S9 mix if required, 0.1 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to give a final volume of 20 ml (R10 is used for the 24-hour exposure group).
-The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
-At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/ml. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 10^5 cells/ml.
-On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.
-The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING
Microtitre plates were scored using a magnifying mirror box after 10 to 14 days’ incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded.
-Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately 0.25 to 0.75 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

CALCULATION OF PERCENTAGE RELATIVE SUSPENSION GROWTH (%RSG)
-The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the %RSG.
-Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
-Day 0 Factor = (dose 0-hour cell count)/(vehicle control 0-hour cell count)
-%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

CALCULATION OF DAY 2 VIABILITY (%V)
-Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
-P(0) = number of negative wells/total wells plated
-%V = - (lnP(0) x 100)/(number of cells/well)

CALCULATION OF RELATIVE TOTAL GROWTH (RTG)
-For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = (%V/Mean Solvent Control %V) x 100%
-Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

CALCULATION OF MUTATION FREQUENCY (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

Evaluation criteria:

INTERPRETATION OF RESULTS
-The normal range for mutant frequency per survivor is 50-170 x 10^-6 for the TK+/- locus in L5178Y cells at the test laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10^-6 mutant frequency per survivor are not normally acceptable and require repetition.
-Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Statistics:

-The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al, 1989).
-For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
-There was evidence of marked dose-related reductions in the %RSG of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult.
-A greasy/oily precipitate of the test item was present at higher concentrations. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.


MUTAGENICITY TEST
The results are summarised in Tables 1 and 2.

Experiment 1
-There was once again evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation.
- There was also evidence of a modest reduction in %V in the presence of metabolic activation, therefore indicating that residual toxicity had occurred.
-Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the presence of metabolic activation. Optimum levels of toxicity were not achieved in the absence of metabolic activation. This was considered to be due to a combination of the steep toxicity curve of the test item, and the presence of greasy/oily precipitate giving variable exposure of the test item to the cells. However, with no evidence of any toxicologically significant increases in mutant frequency in the presence of metabolic activation in Experiment 1, or in the absence of metabolic activation in Experiment 2 where optimum levels of toxicity were achieved, the test item was considered to have been overall adequately tested.
-The excessive toxicity observed at 468.75 µg/ml in the absence of metabolic activation, and at 625 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
-Neither of the vehicle control mutant frequency values were outside the acceptable range. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
-The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in the absence of metabolic activation.
-A very modest statistically significant dose related (linear-trend) increase was observed in the presence of metabolic activation. However, there was no evidence of any marked increases in mutant frequency, the GEF was not exceeded at any of the dose levels, and there was no evidence of any increases in absolute number of mutant colonies. Therefore, the response was considered artefactual and of no toxicological significance.

Experiment 2
-As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values.
-There was no evidence of any significant reductions in %V in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
-Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation. On this occasion optimum levels of toxicity were not achieved in the presence of metabolic activation. This was considered to be due to a combination of the steep toxicity curve of the test item, the presence of greasy/oily precipitate giving variable exposure of the test item to the cells, and the lowering of the S9 concentration resulting in lower levels of toxicity than seen previously. However, with no evidence of any toxicologically significant increases in mutant frequency in the absence of metabolic activation in Experiment 2, or in the presence of metabolic activation in Experiment 1 where optimum levels of toxicity were achieved, the test item was considered to have been overall adequately tested.
-The excessive toxicity observed at 350 µg/ml in the absence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
-The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. It should be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in lower levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.
-Neither of the vehicle control mutant frequency values were outside the acceptable range. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
-The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in either the absence or presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Summary of Results from Experiment 1

Treatment (µg/ml)	4-Hours -S9			Treatment (µg/ml)	4-Hours +S9
	%RSG	RTG	MF*		%RSG	RTG	MF*
0 9.77† 19.53 39.06 78.13 156.25 234.38 312.5 468.75†	100 100 93 95 89 81 76 57 1	1.00   1.03 1.05 0.91 0.85 0.77 0.58	115.33   98.86 84.26 78.54 91.48 85.07 82.63	0 9.77† 19.53 39.06 78.13 156.25 312.5 468.75 625†	100 97 99 92 95 89 67 14 1	1.00   1.08 0.93 0.87 0.84 0.59 0.09	102.83   88.77 119.03 111.70 105.31 138.15 139.82
Linear trend: Not significant				Linear trend: p<0.05
Positive Control EMS 400	69	0.48	802.31	Positive Control CP 2	59	0.30	1320.32

*5-TFT resistant mutants/10^6 viable cells 2 days after treatment

†Not plated for viability or TFT resistance

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

 

Table 2 Summary of Results from Experiment 2

Treatment (µg/ml)	24-Hours -S9			Treatment (µg/ml)	4-Hours +S9
	%RSG	RTG	MF*		%RSG	RTG	MF*
0 6.25† 12.5† 25† 50 100 150 200 250 300 350†	100 109 83 99 93 57 49 37 21 9 5	1.00       0.93 0.64 0.62 0.49 0.35 0.18	176.95       186.78 169.79 159.56 167.58 159.79 97.47	0 50† 100† 200 300 350 400 450 500	100 98 87 81 75 70 64 59 40	1.00     0.90 0.90 0.84 0.80 0.62 0.43	172.76     145.89 159.43 146.53 134.78 171.61 166.75
Linear trend: Not significant				Linear trend: Not significant
Positive Control EMS 150	71	0.49	1836.31	Positive Control CP 2	66	0.46	1355.21

*5-TFT resistant mutants/10^6 viable cells 2 days after treatment

†Not plated for viability or TFT resistance

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

A GLP compliant study was performed according to the standardised guidelines OECD 476, Method B.17 of Commission Regulation (EC) No. 440/2008, US EPA OPPTS 870.5300, and would be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

The study was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, both in the absence and presence of metabolic activation (S9 mix).

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, with or without metabolic activation, in either the first or the second experiment.

Therefore the test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.