Reaction products of petrolatum (petroleum), oxidized, esterified with methanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 2003 to 15 September 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

MHLW and MAFF

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse 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 state: Brown waxy solid
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Histidine auxotrophs of S. typhimurium and tryptophan auxotrophs of E. coil

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: uvrB, rfa

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (10% liver S9 in co-factors)

Test concentrations with justification for top dose:

Preliminary Study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main Study: 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Remarks:

see table 1 in the field "any other information on material and methods inc tables"

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

see table 1 in the field "any other information on material and methods inc tables"

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method

PRELIMINARY STUDY
- The dose range was determined through a preliminary toxicity assay
- Test strains, TA 100 and WP2uvrA
- 0.1 mL of the culture was added to 2 mL of molten top agar (either histidine or tryptophan supplemented), 0.1 mL of the test material and 0.5 mL of S9 mix or phosphate buffer. This was overlaid onto sterile agar plates of Vogel-Bonner Minimal agar (30 mL /plate).
- The solvent control and sterility controls were performed.
- The plates were incubated for 48 hours at 37 ºC.
- Revertant colonies were counted using a Domino colony counter.

MAIN TEST
- The main test was carried out twice on different days, following the same procedure, with fresh bacterial cultures.
- The procedure is the same as in the preliminary test; all plates were produced in triplicate.
- The colonies had to be counted by hand in the 2nd test as there was interference from the agar plates.

Evaluation criteria:

he test was considered positive if the following criteria was met:
The test induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

The mutation assay was considered to be valid if the following criteria were met:
All culture strains in the vehicle and untreated controls exhibit a characteristic number of spontaneous revertants per plate.
Strain characteristics have been confirmed i.e. rfa and pKM101 plasmid R-factor.
All strain cultures meet the approximate range of 1 to 9.9 x 10⁹ bacteria per mL.
Positive controls should be at least two times the respective vehicle control values.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Statistics:

Dunnett's method of linear regression.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments was shown to be sterile.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. A light, particulate precipitate was observed at 5000 pg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 2. Revertant Colony Counts from Experiment 1 and 2 Including Controls

Experiment No.	With or Without S9-mix	Concentration of Test Material	Mean Number of Revertants per Plate (standard deviation)
			Strain
			TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
1	-	0	66 (13.1)	11 (1.0)	22 (6.1)	20 (3.5)	7 (1.2)
		50	70 (6.0)	12 (3.1)	22 (6.1)	19 (3.5)	9 (2.1)
		150	87 (13.2)	13 (3.5)	21 (4.6)	21 (7.5)	8 (0.6)
		500	71 (13.0)	14 (10.1)	15 (1.2)	16 (7.1)	6 (2.6)
		1500	71 (4.4)	13 (1.5)	21 (3.5)	17 (3.2)	8 (0.6)
		5000	72 (13.9)	7 (2.3)	15 (3.5)	20 (5.0)	9 (2.5)
		ENNG	514 (10.4)	275 (16.0)	797 (22.5)
		4NQO				240 (13.1)
		9AA					2316 (390.7)
	+	0	71 (5.6)	13 (5.6)	24 (6.4)	27 (3.6)	15 (5.5)
		50	71 (4.7)	10 (3.8)	18 (3.5)	22 (0.0)	16 (5.0)
		150	68 (5.7)	8 (0.6)	22 (6.6)	23 (3.5)	15 (2.5)
		500	69 (1.2)	11 (3.6)	18 (6.8)	25 (9.9	17 (3.5)
		1500	67 (5.8)	9 (2.3)	20 (0.6)	21 (5.5)	17 (6.7)
		5000	69 (4.7)	14 (4.0)	21 (2.1)	25 (7.8)	10 (3.5)
		2AA	1440 (32.1)	156 (1.5)	791 (16.4)		502 (53.5)
		BP				205 (15.1)
2	-	0	130 (9.5)	35 (3.5)	20 (2.0)	24 (3.8)	12 (3.5)
		50	115 (2.1)	37 (3.5)	23 (5.2)	21 (2.3)	9 (3.2)
		150	129 (14.4)	39 (0.0)	17 (2.3)	23 (5.0)	15 (2.5)
		500	115 (15.7)	31 (6.1)	17 (3.5)	16 (2.6)	13 (1.5)
		1500	99 (13.0)	27 (12.3)	19 (1.5)	22 (1.5)	11 (3.0)
		5000	88 (4.6)	15 (9.5)	18 (4.6)	18 (1.7)	6 (2.1)
		ENNG	381 (11.8)	137 (25.0)	817 (39.1)
		4NQO				210 (9.9)
		9AA					827 (65.5)
	+	0	103 (17.2)	13 (4.4)	29 (2.3)	35 (5.5)	19 (2.1)
		50	115 (9.1)	14 (1.7)	20 (0.0)	34 (4.0)	20 (4.0)
		150	101 (9.8)	15 (2.6)	25 (4.5)	30 (2.1)	18 (2.6)
		500	90 (3.1)	14 (1.5)	22 (2.1)	26 (3.0)	19 (6.7)
		1500	77 (6.7)	13 (1.2)	17 (2.5)	20 (6.6)	15 (2.8)
		5000	89 (11.0)	17 (1.7)	23 (1.5)	13 (0.6)	19 (1.2)
		2AA	881 (50.6)	229 (59.7)	613 (64.6)		316 (40.8)
		BP				260 (20.2)

Applicant's summary and conclusion

Conclusions:

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

Under the conditions of the test, the test material was determined to be non mutagenic in several strains of S. typhimurium and E. coli confirmed in an Ames 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 WP2 uvr A were exposed to the test material in varying concentrations with and without metabolic activation. Under the conditions of the test it was determined that the test material is non-mutagenic as there was no significant increase in revertant colony counts.