Paraffin waxes and Hydrocarbon waxes, oxidized

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see read-across justifocation in section Chapter 13

Reason / purpose for cross-reference:

read-across source

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'.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: Mouse Lymphoma Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2nd January 2014 to 10th April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The following culture media were used; minimal medium A, minimal medium B, complete medium (5 %), complete medium (10 %), complete medium A (20 %) and complete medium B (20 %). The compositions of media can be seen in Table 1 in the field “any other information on materials and methods incl. tables”.
- Properly maintained: Yes, Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically "cleansed" against high spontaneous background: Yes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Main Assay I (with and without metabolic activation): 6.25, 12.5, 25.0, 50.0 and 100 µg/mL
Main Assay II (without metabolic activation): 6.25, 12.5, 25.0, 50.0 and 100 µg/mL
Main Assay II (with metabolic activation): 19.8, 29.6, 44.4, 66.7 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. A cell suspension (1 x 10^6 cells/mL) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes. The treatment media were prepared as follows, with a total volume of 20 mL:
-3 hour exposure time, without S9 metabolism: 10.0 mL of cell suspension (1 x 10^6 cells/mL in complete medium 5 %), 9.8 mL complete medium (5 %) and 0.2 mL of the control or appropriate test solution.
-24 hour exposure time, without S9 metabolism: 3.0 mL of cell suspension (1 x 10^6 cells/mL in complete medium 10 %), 16.8 mL complete medium (10 %), and 0.2 mL of the control or appropriate test solution.
-3 hour exposure time, with S9 metabolic activation: 10.0 mL of cell suspension (1 x 10^6 cells/mL in complete medium 5 %), 9.8 mL S9 mix, and 0.2 mL of the control or appropriate test solution.
The cultures were incubated with the test solution at 37 °C. At the end of the exposure period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).
After washing cells were resuspended in fresh complete medium (10 %) and cell densities were determined. The number of cells was adjusted to give 2 x 10^5 cells/mL.
The cultures were incubated at 37 °C in a 5 % CO2 atmosphere (100 % nominal relative humidity) to allow for expression of the mutant phenotype.

Determination of Survival
Following adjustment of the cell densities, samples of the cultures were diluted to 8 cell/mL using complete medium A (20 %). A 0.2 mL aliquot of each diluted culture was placed into each well of two 96-well plates. The plates were incubated at 37 °C in a 5 % CO2 atmosphere (100 % nominal relative humidity) for 6 - 9 days. After incubation, wells containing viable clones were identified by the eye using background illumination and then counted.

Expression Period
During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2 x 10^5 cells/mL.

Plating for 5-Trifluorothymidine Resistance
After dilution, the cell suspensions in complete medium B (20 %) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 x 10^3 cells were plated in each well of four 96-well plates.
Plates were incubated at 37 °C in a 5 % CO2 atmosphere (100 % nominal relative humidity) for 12 - 13 days and wells containing clones were identified as described above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored.

Plating for Viability
After dilution, in complete medium A (20 %), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37 °C in a 5 % CO2 atmosphere (100 % nominal relative humidity) for 12 - 13 days and wells containing clones were identified as above and counted.

DURATION
- Exposure duration:
> Main Assay I:
Assay 1: 3 hours, with and without metabolic activation.
>Main Assay II:
Assay 2: 24 hours, without metabolic activation.
Assay 3: 3 hours, with metabolic activation.
- Expression time (cells in growth medium): 6 to 9 days.

NUMBER OF REPLICATIONS: Duplicate cultures were prepared for each test concentration, with the exception of the positive controls which were prepared in a single culture.

DETERMINATION OF CYTOTOXICITY
- Method: Relative survival value, cell count factors, raw plate counts.

PRELIMINARY TEST
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item were used and the survival of the cells was subsequently determined.
Treatments wee performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic aqctivation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS) , cells were resuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose elvel, 0.2 mL was plated into 96 microtitre wells. The plates were incubated at 37 degs. C in a 5% CO2 atmosphere (100% relative humidity) for 7 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1) The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126 x 10^-6) at one or more doses.
2) There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990). The following were calculated:
- Test for consistency between plates
- Heterogeneity factors for replicate cultures
- Test for overall consistency
- Updated heterogeneity factors
- Comparison of each treatment with the control
- Test for linear trend

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Main Assay I; with 3 hour exposure with S9 at 50.0 and 100 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality and pH: Addition of the test solution did not have any obvious effect on the osmolality or pH of the treatment medium.
- Precipitation:
> In Main Assay I, in the absence of S9 metabollic activation, higher than usual heterogenicity was observed in plate counts for mutation between Culture B replicate plate at 12.5 µg/mL and between replicate cultures at 50 µg/mL. Because of the inclusion of this last dose in analysis, the overall heterogeneity between cultures was higher than usual. Since this was probably due to the presence of test item precipitation in the treatment mixture, it was not considered to have affected the validity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 100 µg/mL and at a wide range of lower dose levels: 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 µg/mL. Upon addition of the test items to the cultures, in the absence of S9 metabolism, a slight cloudy appearance of the treatment medium was noted at the two highest concentrations. At the end of the 3 hour treat period, a dose-related cloudy appearance was observed starting from 12.5 µg/mL, both in the absence and presence of S9 metabolic activation; while by the end of the 24 hour treat period, no opacity was noted.

In the absence of S9 metabolic activation, using both the 3 and 24 hour treatment times, no toxicity was observed at any concentration tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), slight toxicity was seen at the two highest concentrations (53 and 63% RS), while no relevant toxicity was observed over the remaining dose levels tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 2: Summary of Main Assay I

Dose Level (µg/mL)	RS (%)	RTG (%)	MFØ	P	IMFØ	Proportion Small Colony Mutants	Precipitation
3 Hours Without S9
0	100	100	79.5	-	-	0.43	-
6.25	125	141	50.0	NS	-	-	-
12.5	117	115	57.9	NS	-	-	-
25.0	127	130	71.4	NS	-	-	+
50.0	129	133	77.0	NS	-	-	+
100	129	119	89.0	NS	9.56	-	+
MMS (10.0)	129	93	326.7	-	247.3**	0.49	-
Linear Trend				*
3 Hours With S9
0	100	100	93.3	-	-	0.27	-
6.25	88	98	83.5	NS	-	-	-
12.5	112	102	95.4	NS	2.07	-	-
25.0	143	104	55.0	NS	-	-	+
50.0	64	59	94.5	NS	1.13	-	+
100	71	62	67.8	NS	-	-	+
B(a)P (2.00)	42	18	1141.9	-	1048.5**	0.54	-
Linear Trend				NS

 

Table 3: Summary of Main Assay II

Dose Level (µg/mL)	RS (%)	RTG (%)	MFØ	P	IMFØ	Proportion Small Colony Mutants	Precipitation
24 Hours Without S9
0	100	100	60.3	-	-	0.34	-
6.25	86	98	61.1	NS	0.82	-	-
12.5	80	114	43.6	NS	-	-	-
25.0	97	96	59.2	NS	-	-	-
50.0	86	103	61.3	NS	0.98	-	+
100	90	97	66.3	NS	6.01	-	+
MMS (5.0)	68	78	299.3	-	239**	0.34	-
Linear Trend				NS
3 Hours With S9
0	100	100	67.6	-	-	0.30	-
19.8	85	94	89.3	NS	21.71	-	-
29.6	95	110	64.9	NS	-	-	-
44.4	99	112	70.5	NS	2.89	-	+
66.7	85	84	104.9	NS	37.26	-	+
100	91	103	92.1	NS	24.53	-	+
B(a)P (2.00)	34	29	613.6	-	546**	0.38	-
Linear Trend				*

Ø = per 10^6 viable cells

NS = Not statistically significant

* = Statistically significant at p < 5%

** = Induced mutant frequency (IMF) > global evaluation factor (GEF = 126 x 10^-6)

+ = Opacity of treatment medium

RS = Relative survival

RTG = Relative total growth

MF = Mutation frequency

IMF = Induced mutant frequency

MMS = methylmethanesulphonate

B(a)P = benzo(a)pyrene

Table 4: Historical Data, Mutation Frequency per Million Surviving Cells

	Without S9, 3 hours		With S9, 3 hours		Without S9, 24 hours
	Negative Control	Positive Control	Negative Control	Positive Control	Negative Control	Positive Control
Mean	88.0	446	90.8	694	85.1	546
SD	28.6	148	30.2	267	30.5	203
Upper Confidence Limit (P<1%)	162	NC	169	NC	164	NC
Min	51	209	44.9	187	50.4	221
Max	234	1103	234	1393	217	1950
n	90	119	196	196	85	85

NC = not calculated; n = number of experiments.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the study, the test material did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation.

Executive summary:

In a GLP compliant study performed to standardised guidelines OECD 476 and EU Method B.17, the mutagenic potential of the test material was evaluated in a mouse lymphoma assay. Mouse lymphoma L5178Y cells were exposed to the test material in two experiments at concentrations ranging from 6.25 to 100 µg/plate, both in the presence and absence of metabolic activation. Main Assay I was performed with an exposure time of 3 hours with and without metabolic activation. Main Assay II was performed for 3 hour with metabolic activation and for 24 hours without.

No mutant frequency above those seen in the concurrent solvent control or historical background ranges were observed following treatment with the test material, in the absence or presence of S9 metabolism.

It was concluded that under the conditions of the study, the test material did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: Ames-Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2008 to 13 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2A Genotoxicity: Specific Aspects of Regulatory Tests, Step 5.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan operons in S. typhimurium and E. coli, respectively.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Type and identity of media:
- Nutrient Broth: Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5 % in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
- Nutrient Agar: Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
- Minimal Agar: Minimal medium agar was prepared as 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E, with 2 % Glucose, and poured into 9 cm plastic Petri dishes.
- Top Agar: "Top Agar" (overlay agar) was prepared as 0.6 % Difco Bacto-agar + 0.5 % NaCl in distilled water. Prior to use 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 mL).
- Properly maintained: Yes, permanent stocks of these strains are kept at -80 °C. Overnight subcultures of these stocks were prepared for each day's work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
> Histidine requirement: No Growth on Minimal plates + Biotin and Growth on Minimal plates + Biotin + Histidine.
> Tryptophan requirement: No Growth on Minimal agar plates and Growth on Minimal plates + Tryptophan.
> uvrA, uvrB: Sensitivity to UV irradiation.
> rfa: Sensitivity to Crystal Violet.
> pKM101: Resistance to Ampicillin.

Additional strain / cell type characteristics:

other: uvrA, uvrB, rfa and pKM101.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Main Assay I: 62.5, 125, 250, 500 and 1000 µg/plate
Main Assay II: 31.3, 62.5, 125, 250 and 500 µg/plate
Preliminary toxicity test: 0.0, 10.0, 31.6, 100, 316 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

MAIN ASSAY I:
METHOD OF APPLICATION: in agar (plate incorporation). The components of the assay (0.1 mL of the tester strain bacteria, 0.1 mL of the test or control solution and 0.5 mL of the S9 mix or phosphate buffer (pH 7.4, 0.1 M)) were added to 2 mL of molten overlay agar (held at 45 °C) and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate, and allowed to solidify prior to incubation.

MAIN ASSAY II:
METHOD OF APPLICATION: pre-incubation method. The following components were added in turn to an empty test-tube as follows: 0.1 mL of bacterial suspension, 0.05 mL of the appropriate test or control solution and 0.5 mL of S9 mix or phosphate buffer (pH 7.4, 0.1 M) as necessary. The incubate was vortexed and placed at 37 °C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

DURATION
- Preincubation period: 30 minutes for Main Assay II.
- Exposure duration: 72 hours, incubated at 37 °C.

NUMBER OF REPLICATIONS: Three.

SCORING: counting the number of revertant colonies on each plate.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

OTHER EXAMINATIONS:
- Sterility check: Performed on the S9 mix, test solutions and bacterial cultures. Each substance was plated on nutrient agar plates to establish the number of bacteria in the cultures.

PRELIMINARY TOXICITY TEST:
- A preliminary toxicity test was performed in the presence and absence of metabolic activation using the plate incorporation method, a DMSO (100 µL) vehicle control was run concurrently. A single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. The results were used to determine the concentration levels for the definitive test.
- Precipitation: Precipitation was observed at the end of the incubation period at the two highest dose levels, 316 and 1000 µg/plate. This was, however, not considered to have interfered with the scoring of the revertant colonies.
- Toxicity: No toxicity was observed at any of the dose levels with any of the tester strains, in the absence or presence of metabolic activation.

Evaluation criteria:

For the test material to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The mean and standard deviation for results from Main Assays I and II were calculated.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the end of the incubation period, in both Main Assays I and II. This was not considered to have interfered with the scoring of the revertant colonies.
> Main Assay I: precipitation at 1000 and 500 µg/plate.
> Main Assay II: precipitation at 500, 250 and 125 µg/plate.
- Other confounding effects: No plates were lost through contamination or cracking.

COMPARISON WITH HISTORICAL CONTROL DATA: Results show that mean plate counts for untreated and positive control plates fell within the normal historical range. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 millions for each strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity: No toxicity was observed at any of the dose levels with any of the tester strains, in the absence or presence of metabolic activation for both Main Assays I and II.

STERILITY CONTROL
The sterility of the S9 mix and the test solutions were confirmed by the absence of colonies on additional agar plates spread separately with these solutions.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Main Assay I

Table 1: Preliminary Toxicity Test, Number of Revertant Colonies per Plate

Dose Level (µg/plate)	Tester Strains Without Metabolic Activation					Tester Strains With Metabolic Activation
	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Untreated	19	17	27	175	20	10	18	36	181	29
DMSO 100 µL	17	23	30	174	22	13	20	35	178	28
10.0	23	23	37	152	30	21	16	42	166	28
31.6	18	17	30	168	25	18	19	42	152	29
100	16	15	36	148	27	13	21	39	142	34
316	25 p	15 p	35 p	163 p	24 p	19 p	18 p	39 p	170 p	32 p
1000	17 p	23 p	39 p	165 p	26 p	23 p	17 p	36 p	165 p	30 p

p = precipitation

 

Table 2: Main Assay I, Mean Number of Revertant Colonies per Plate (± SD)

Dose Level (µg/plate)	Tester Strains Without Metabolic Activation					Tester Strains With Metabolic Activation
	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Untreated	16 (0.3)	17 (0.3)	29 (0.9)	157 (22.5)	27 (1.2)	18 (1.2)	22 (1.5)	36 (1.0)	148 (10.2)	32 (0.9)
0.00	19 (0.3)	17 (1.5)	25 (1.0)	175 (11.3)	26 (0.6)	17 (1.3)	18 (0.6)	40 (1.5)	148 (19.5)	29 (0.9)
62.5	18 (1.2)	16 (2.0)	31 (1.8)	144 (26.5)	29 (3.0)	15 (1.3)	14 (2.1)	34 (0.6)	166 (12.8)	30 (2.3)
125	18 (1.2)	14 (0.9)	29 (0.9)	156 (16.5)	31 (2.6)	19 (0.6)	14 (1.9)	35 (1.3)	167 (17.2)	29 (0.3)
250	19 (2.0)	14 (2.0)	28 (2.1)	164 (13.0)	29 (2.3)	22 (1.2)	17 (0.9)	33 (0.9)	140 (6.6)	32 (1.5)
500	16 (2.0) p	18 (1.5) p	27 (1.9) p	127 (9.6) p	33 (2.2) p	18 (1.5) p	16 (1.7) p	31 (2.6) p	137 (9.5) p	28 (0.9) p
1000	15 (1.5) p	14 (2.1) p	21 (2.6) p	145 (14.9) p	23 (0.9) p	18 (1.5) p	14 (1.3) p	29 (1.8) p	140 (5.2) p	29 (0.9) p

p = precipitation

 

Table 3: Main Assay I, Mean Control Number of Revertant Colonies per Plate (± SD)

Tester Strain		Without Metabolic Activation						With Metabolic Activation
		Untreated	SA	DMSO	9-AA	MMS	2-N	DMSO	2-AA
TA 1535	Mean (± SD)	16 (0.3)	544 (27.9)	-	-	-	-	17 (1.3)	136 (7.8)
TA 1537	Mean (± SD)	-	-	17 (1.5)	160 (5.5)	-	-	18 (0.6)	131 (9.5)
TA 98	Mean (± SD)	-	-	25 (1.0)	-	-	177 (6.1)	40 (1.5)	555 (27.2)
TA 100	Mean (± SD)	157 (22.5)	626 (10.4)	-	-	-	-	148 (19.5)	1021 (41.2)
WP2uvrA	Mean (± SD)	27 (1.2)	-	-	-	150 (7.1)	-	29 (0.9)	230 (2.9)

SA = Sodium Azide (1 µg/plate); DMSO = dimethylsulfoxide (100 µL/plate); 2-AA = 2-Aminoanthracene (1 µg/plate or 10 µg/plate for WP2uvrA); 9-AA = 9-Aminoacridine (50 µg/plate); MMS = methylmethanesulfonate (500 µg/plate); 2-N = 2-Nitrofluorene (2 µg/plate).

 

Table 4: Main Assay II, Mean Number of Revertant Colonies per Plate (± SD)

Dose Level (µg/plate)	Tester Strains Without Metabolic Activation					Tester Strains With Metabolic Activation
	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Untreated	19 (0.9)	17 (0.3)	29 (1.5)	162 (13.0)	27 (1.5)	18 (1.0)	20 (0.9)	41 (0.9)	153 (13.8)	32 (1.2)
0.00	15 (2.3)	17 (1.2)	28 (1.5)	145 (10.2)	24 (3.1)	20 (0.7)	18 (0.9)	41 (1.9)	162 (6.1)	34 (0.9)
31.3	14(2.2)	18 (0.7)	29 (1.3)	121 (5.5)	27 (1.0)	20 (0.7)	19 (1.5)	42 (1.9)	175 (6.4)	39 (1.0)
62.5	13 (0.6)	22 (1.3)	28 (2.0)	171 (6.4)	29 (3.5)	15 (1.0)	18 (0.3)	34 (01.5)	173 (4.7)	36 (1.8)
125	15 (1.8) p	18 (1.2) p	29 (1.2) p	154 (8.0) p	26 (2.0) p	20 (2.0) p	20 (3.2) p	37 (2.4) p	179 (8.1) p	40 (0.9) p
250	17 (1.2) p	15 (1.3) p	25 (1.8) p	132 (7.3) p	28 (2.3) p	19 (2.3) p	20 (0.9) p	38 (2.7) p	183 (4.2) p	32 (1.2) p
500	18 (1.8) p	16 (1.9) p	29 (1.3) p	140 (11.6) p	29 (1.2) p	23 (0.9) p	21 (1.2) p	36 (0.3) p	172 (2.3) p	33 (3.5) p

p = precipitation

 

Table 5: Main Assay II, Mean Control Number of Revertant Colonies per Plate (± SD)

Tester Strain		Without Metabolic Activation						With Metabolic Activation
		Untreated	SA	DMSO	9-AA	MMS	2-N	DMSO	2-AA
TA 1535	Mean (± SD)	16 (2.1)	465 (16.4)	-	-	-	-	20 (1.7)	114 (6.1)
TA 1537	Mean (± SD)	-	-	20 (1.2)	153 (18.3)	-	-	25 (1.5)	122 (2.7)
TA 98	Mean (± SD)	-	-	26 (1.10)	-	-	180 (3.4)	37 (0.9)	545 (23.4)
TA 100	Mean (± SD)	125 (4.7)	532 (24.2)	-	-	-	-	133 (5.1)	1177 (37.9)
WP2uvrA	Mean (± SD)	25 (1.5)	-	-	-	192 (4.0)	-	33 (1.8)	235 (17.9)

SA = Sodium Azide (1 µg/plate); DMSO = dimethylsulfoxide (50 µL/plate); 2-AA = 2-Aminoanthracene (1 µg/plate or 20 µg/plate for WP2uvrA); 9-AA = 9-Aminoacridine (50 µg/plate); MMS = methylmethanesulfonate (500 µg/plate); 2-N = 2-Nitrofluorene (2 µg/plate).

 

Table 6: Historical Data Without Metabolic Activation

		Untreated Plate Incorporation	Untreated Pre-incubation	Positive Control Plate Incorporation	Positive Control Pre-incubation
TA 1535	Mean	18	18	570	571
	SD	2.6	2.4	77.2	74.6
	n	164	168	164	168
TA 1537	Mean	17	17	169	163
	SD	2.0	1.8	34.4	42.9
	n	163	168	163	168
TA 98	Mean	31	32	185	180
	SD	3.2	2.9	25.0	24.4
	n	166	160	166	160
TA 100	Mean	143	141	796	804
	SD	15.6	15.2	121.2	118.8
	n	166	165	166	165
WP2uvrA	Mean	28	28	181	212
	SD	3.2	3.0	18.2	45.1
	n	108	103	108	103

 

Table 7: Historical Data With Metabolic Activation

		Untreated Plate Incorporation	Untreated Pre-incubation	Positive Control Plate Incorporation	Positive Control Pre-incubation
TA 1535	Mean	16	16	142	94
	SD	1.7	1.8	29.0	9.9
	n	164	167	164	167
TA 1537	Mean	21	21	121	97
	SD	2.5	2.1	20.5	11.5
	n	163	169	163	169
TA 98	Mean	39	40	621	668
	SD	3.8	2.9	141.3	186.1
	n	166	150	166	150
TA 100	Mean	152	149	1282	1144
	SD	16.1	14.6	193.8	189.6
	n	167	165	167	165
WP2uvrA	Mean	35	34	203	190
	SD	3.4	3.1	36.6	34.5
	n	107	98	107	98

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test material did not induce a two-fold increase in the number of revertant colonies, at any dose level up to the maximum concentration of 1000 µg/plate, in any tester strain, in the absence or presence of metabolic activation. It was therefore concluded that the test material is not mutagenic to S. typhimurium or E. coli under the conditions of the test.

Executive summary:

In a GLP compliant study performed to standardised guidelines OECD 471 and EU Method B.13/14, the mutagenic potential of the test material was assayed in an Ames Test. S. typhimurium TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2uvrA were exposed to the test material in two experiments at concentrations ranging from 62.5 to 1000 µg/plate using the plate incorporation method and 31.3 to 500 µg/plate using the pre-incubation method. Both tests were performed with and without metabolic activation.

The test material did not induce an increase in the number of revertant colonies, at any dose level up to the maximum concentration of 1000 µg/plate, in any tester strain, in the absence or presence of metabolic activation. No toxicity was observed at any of the dose levels with any of the tester strains, in the absence or presence of metabolic activation for both Main Assays I and II. Precipitation was observed at the end of the incubation period, in Main Assay I ≥ 500 µg/plate and in Main Assay II ≥ 125 µg/plate. This was not considered to have interfered with the scoring of the revertant colonies. It was therefore concluded that under the conditions of the test, the test material is not mutagenic to S. typhimurium or E. coli.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The key studies used in the assessment of the genotoxicity of the registered substance have been assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch (1997). These studies,and all the associated supporting studies, which include bacterial reverse mutation, chromosomal aberration and mouse lymphoma assays, indicate that the registered substance does not have a potential to induce genetic toxicity.

The chromosome abberation

Oxidized wax, lithium salt was evaluated for its chromosomal abberation potential in chinese hamster ovary cells accirding to a standardized, OECD 473 Chromosomal abberation study protocol (Wright, 2004) that has been been designated as the key study for this endpoint and given a rating of 1 according to the Klimich criteria. 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.

Bactrial reverse mutation (Ames assay)

The key Ames tests of the lithium salt of oxidized wax (Cinelli, 2008) and supporting Ames tests of similar substances have also been included (Cinelli, 2008a and Anonymus 2000a), the results of which were both in agreement with the key studies and were assigned a Klimisch score of 1. In all of these studies the test material was administered to five bacterial strains,S. typhimuriumTA 1535, TA 1537, TA 98, TA 100 andE. coliWP2uvrA, in the presence and absence of metabolic activation using both the plate incorporation and pre-incubation methods. Under the conditions of each test the test material was found to be non-mutagenic in all strains tested.

Mouse lymphoma

A mouse lymphoma study of the lithium salt of oxidized wax (Bisini, 2014) has been provided as key information. Under conditions of the test, the test material did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation. This study was performed to OECD and EU guidelines under GLP conditions and was assigned a Klimisch score of 1.

Justification for classification or non-classification

None of the studies conducted on oxidized wax lithium salt included in this evaluation produced results that met the criteria for classification for genotoxicity.