Paraffin waxes and Hydrocarbon waxes

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2005-06-17 to 2005-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restriction by the because it was carried out in accordance with OECD Test Guideline 471 and is GLP compliant.

Justification for type of information:

The standard OECD 471 test is not suitable to test petroleum UVCBs, because it has a tendency to produce false negatives for these substances. Therefore, the petroleum industry has developed a Modified Ames assay, optimized to accurately identify positive results for this endpoint. This deviation from the prescribed testing procedure requires some further explanation which is given in the attached document. The document gives a brief history of the development of the Modified Ames test and outlines Concawe’s proposed work (as part of a wider testing strategy, see Annex 13) to further support the use of this test for PS.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report Date:

2005

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Test material form:

liquid: viscous

Details on test material:

-Test Substance: CAS number: 8002-74-2 (100% purity)
Chemical name: aliphatic alkane
Other name: hydrocarbon wax
Appearance: solid, white, waxy
Batch/Lot Number: A053603
Molecular Formula: C(n)H(2n+2); Sasolwax 5203 contains alkanes ranging from C19 to C36. The man component is C26
Average Molecular Weight: 360
Melting point: 54°C
TNO Test Substance Number: 0500A9

Prior to administration, test substance was melted and extracted in DMSO at a concentration of 50 mg/ml Sasolwax 5203 for approximately 30 minutes at 60±2°C under agitation. After 30 minutes incubation, the wax was coagulated by incubation at 6°C for 8 minutes. The extract (DMSO aliquot) was collected. The test substance was melted again and extraction procedure was repeated. A light white extraction solution was obtained. The extracts were pooled and serial dilutions, separated by 3-fold intervals, in DMSO were prepared.

- Viscosity (3.5 mm2/s at 100 °C)
- Congealing point (54 °C)
- Color saybolt ASTM D156 (30)
- Oil content (0.2%)

Method

Target gene:

No data reported.

Species / strain

Species / strain / cell type:

other: S. typhimurium TA98, 100, 1535, 1537 and E. coli WP 2 uvrA

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Wistar rat S9 homogenate

Test concentrations with justification for top dose:

Just before use, the test substance was extracted with DMSO at 50 mg/ml. The test substance was melted at approximately 60°C and extracted with half of the volume of vehicle needed, for 30 minutes at 60 ± 5°C under agitation. After the 30 minutes incubation, the wax was coagulated by incubation at ± 6°C for ± 8 minutes. The extract (DMSO aliquot) was collected. The test substance was melted again and extraction procedure was repeated. A light white extraction solution was obtained. The extracts were pooled and serial dilutions, separated by 3-fold intervals, in DMSO were prepared. A highest dose 100 % extract was tested. In total test five dose levels, ranging from 1.23 to 100 % of the extract were tested, which is comparable to 62 to 5000 μg of the test substance per plate, assuming a complete extraction. Nominal concentrations were used.

Vehicle / solvent:

dimethyl sulphoxide

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: In the absence of S9, positive controls were sodium azide 1.0 µg/plate, 9-aminoacridine 80.0 µg/plate, 2-nitrofluorene 2.0 µg/plate, N-ethyl-N-nitrosourea 100 µg/plate. In the presence of the S9 mix, 2-aminoanthracene 2.0 µg/plate, benzo(a)pyrene 4.0 µg

Details on test system and experimental conditions:

Briefly, the mutagenicity assay was carried out as follows. To 2 millilitres molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine HCl/0.05 mM biotin for the S. typhimurium strains, and supplemented with 0.05 mM tryptophane for the E. coli WP2 uvrA strain), maintained at approximately 46°C, were added subsequently: 0.1 millilitres of a fully grown culture of the appropriate strain, 0.1 millilitres of the test substance extract, or of the negative or the positive control substance solution, and 0.5 millilitres S9-mix for the experiments with metabolic activation or 0.5 millilitres sodium phosphate 100 millimolar (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed, and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at approximately 37°C for approximately 72 hours. Subsequently, the his+ and trp+ revertants were counted. Cytotoxicity is defined as a reduction (at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.

Evaluation criteria:

The mutagenicity study is considered valid if the mean colony counts of the control values of the strains were within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5% of the plates are lost through contamination or other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if there is a concentration-related increase in the mean number of revertants colonies on the test plates of if a reproducible two-fold or more increase is observed to that on the negative
control plates.

A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Statistics:

No statistic analyses were performed.

Results and discussion

Test results

Species / strain:

other: S. typhimurium strains TA 1535, TA 1537, TA 98, and TA 100; E. coli strain WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Extract of Sasolwax 5203 was slightly toxic, in the presence and absence of S9-mix, to TA 1537 at and above 33.3% of the extract, and to TA 98 at 100% of the extract, as was evidenced by a slightly less dense background lawn compared to the negative control. In both the absence and presence of S9-mix in all strains, the extract of Sasolwax 5203 did not cause a dose-related or more than two-fold increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control. The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Dose (% of extract)	TA 1535		TA 1537		TA 98		TA 100		E. coli
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0%	24	19	2	16	26	47	118	133	44	38
	25	20	13	17	34	42	142	144	38	42
	24	17	19	30	34	49	120	133	43	41
Mean	24	19	11	21	31	46	127	137	42	40
StDev	1	2	9	8	5	4	13	6	3	2
1.23%	34	16	17	22	34	38	103	130	35	38
	26	24	17	28	28	46	143	136	34	32
	25	25	***	24	28	50	118	144	29	40
Mean	28	22	17	25	30	45	121	137	33	37
StDev	5	5	0	3	3	6	20	7	3	4
3.70%	23	13	12	20	28	50	100	137	28	42
	23	18	13	25	37	44	115	150	34	44
	22	19	22	25	37	74	126	124	36	53
Mean	23	17	16	23	34	56	114	137	33	46
StDev	1	3	6	3	5	16	13	13	4	6
11.10%	24	16	10	20	31	37	129	154	35	50
	25	11	16	34	37	53	121	154	40	43
	32	18	12	14	43	40	107	150	38	36
Mean	27	15	13	23	37	43	119	153	38	43
StDev	4	4	3	10	6	9	11	2	3	7
33.30%	26	14	11	35	36	36	138	138	32	34
	26	18	13	12	24	49	125	101	38	34
	22	13	22	19	34	56	137	148	37	43
Mean	25	15	15	22	31	47	133	129	36	37
StDev	2	3	6	12	6	10	7	25	3	5
100%	30	16	7	20	28	41	151	121	35	37
	25	14	8	24	32	50	129	109	31	40
	29	13	13	13	30	55	151	136	44	34
Mean	28	14	9	19	30	49	144	122	37	37
StDev	3	2	3	6	2	7	13	14	7	3
Pos. Control	527	402	1631	165	889	730	728	1493	***	921
	530	452	3461	159	***	691	722	1517	288	1129
	539	380	3266	156	1086	500	694	1569	333	1058
Mean	532	411	2786	160	988	640	715	1526	311	1036
StDev	6	37	1005	5	139	123	18	39	32	106

Mean: Average number of revertants per plate (bolded)

StDev: Standard deviation

S9: Liver homogenate from rats treated with aroclor

Pos. Control Positive control

*** Not counted due to contamination

Underlined values have slightly less dense background lawn of bacterial

Applicant's summary and conclusion

Conclusions:

It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the extract of Sasolwax 5203 is not mutagenic under the conditions employed in this study.

Executive summary:

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 1535, TA 1537, TA 98, TA 100) andE. coli (WP 2 uvrA) were exposed to extracted Sasolwax 5203 in DMSO.  A highest dose of 100% extract was tested. In total, five dose levels ranging from 1.23 to 100% of the extract were tested, which is comparable to nominal concentrations of 62 to 5000 μg of the test substance per plate.  The positive controls induced the appropriate response in the corresponding strains.  There was no evidence of induced mutant colonies over background.

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was carried out in accordance with OECD Test Guideline 471 and is GLP compliant.