Alkenes, C15-18 α-, sulfurized

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:

30 April 1996 to 13 August 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Alkenes, C15-18 α-, sulfurized

EC Number:

267-023-8

EC Name:

Alkenes, C15-18 α-, sulfurized

Cas Number:

67762-55-4

Molecular formula:

Not applicable, complex UVCB

IUPAC Name:

Alkenes, C15-18 α-, sulfurized

Test material form:

other: liquid (unspecified)

Details on test material:

- Appearance: dark liquid
- Storage conditions: room temperature in a clear glass container

Method

Target gene:

Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth #2.
- Properly maintained: yes, stored frozen. All tester strains were checked for the presence of the appropriate genetic markers on approximately a monthly basis.

Additional strain / cell type characteristics:

other: All strains contain an rfa mutation and all except TA102 contain a uvrB deletion mutation. In addition, strains TA98, TA100 and TA102 contain the plasmid pKM101.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth #2.
- Properly maintained: yes, stored frozen. All tester strains were checked for the presence of the appropriate genetic markers on approximately a monthly basis.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (6 % S9 in standard co-factors)

Test concentrations with justification for top dose:

Preliminary Toxicity Test:
0, 50, 167, 500, 1670 and 5000 µg/plate

Mutation Test:
Experiments 1 and 2: 0, 50, 167, 500, 1670, 5000 and 10 000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: isocetyl alcohol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

isocetyl alcohol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

mitomycin C

other: 2-aminofluorene, 2-anthramine

Details on test system and experimental conditions:

- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
2 mL of molten top agar (supplemented with 0.5 mM histidine or tryptophan/0.5 mM biotin) was combined with 0.1 mL tester strain and 0.1 mL of the appropriate concentration of the test material or solvent in sterile glass tubes. Cultures treated in the presence of metabolic activation also contained 0.5 mL of the S9 mixture. The tubes were vortexed and the mixture was poured onto minimal glucose plates, evenly distributed and allowed to solidify. Within an hour all plates were inverted and incubated in the dark.

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: The tests were performed in triplicate.


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
In the absence of metabolic activation, 0.5 mL 0.2 M Na₂HPO₄ (pH 7.4), 0.1 mL tester strain and 0.1 mL of the appropriate concentration of the test material or solvent first were combined and incubated, with shaking, at approximately 30 °C for approximately 30 minutes.
In the presence of metabolic activation, 0.5 mL S9 mixture, 0.1 mL tester strain and 0.1 mL of the appropriate concentration of the test material or solvent first were combined and incubated, with shaking, at approximately 30 °C for approximately 30 minutes.
Following the 30-minute pre-incubation period, 2 mL of molten, supplemented top agar was added to each tube. The tubes were vortexed and the mixture was poured onto minimal glucose plates, evenly distributed and allowed to solidify. Within an hour all plates were inverted and incubated in the dark.

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: The tests were performed in triplicate.


DETERMINATION OF CYTOTOXICITY
- Method: Inhibited growth was characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies.

Evaluation criteria:

A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the solvent control value. Statistical analyses are performed using the program developed by Snee and Irr (1981), with significance established at the 95 % confidence limit. If the test material does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine or tryptophan-independent revertants.

Statistics:

Statistical analyses are performed only when a 50 % increase in revertant frequency, relative to the concurrent negative controls, is observed. As this was not the case for the test material, no analysis took place.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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:

S. typhimurium TA 1537

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 3

Species / strain:

S. typhimurium TA 98

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 4

Species / strain:

S. typhimurium 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 5

Species / strain:

S. typhimurium TA 102

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 6

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:

RANGE-FINDING/SCREENING STUDIES
Results indicated that the test material was not toxic to any strain at any dose evaluated under either treatment condition. In addition, the test material was found to be incompletely soluble (droplets were observed) at all doses.

BACTERIAL CONTAMINANT EVALUATION
To ensure sterility, standard contamination evaluations were performed with each assay. The solvent, S9 mix and highest dose of the test material were evaluated at the same volumes used in the assay. Both top agars also were plated alone on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours at 37°C and then scored for bacterial growth.

MUTAGENICITY EXPERIMENTS
Six doses of the test material were evaluated in the event of insolubility at the highest dose levels. Normal growth was observed in all tester strains at all doses evaluated with and without S9, and the test material was found to be incompletely soluble at all doses (droplets were seen), under both treatment conditions.
Revertant frequencies for all doses in all tester strains with and without S9 under both treatment conditions approximated or were less than those observed in the concurrent negative control cultures.
All positive and negative control values were within acceptable ranges.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type				Frameshift Type
		TA100	TA1535	TA102	WP2uvrA	TA98	TA1537
- - - - - - -	Solvent 50 167 500 1670 5000 10 000	96 67 96 113 108 100 104	7 7 7 9 6 6 10	292 294 292 275 277 269 290	5 7 3 5 4 5 7	26 30 30 27 27 25 23	10 9 12 11 9 11 12
+ + + + + + +	Solvent 50 167 500 1670 5000 10 000	98 119 112 100 101 95 101	10 6 5 5 8 5 9	290 300 309 289 311 297 307	7 6 6 4 5 5 8	35 35 25 30 38 36 31	10 10 12 11 8 10 13
		Positive Controls
-	Name	SA	SA	MMC	ENNG	2NF	9AA
	Concentration (µg/plate)	10	10	2.5	2	5	150
	Mean no. colonies/plate	1014	1109	1599	158	422	947
+	Name	2AM	2AM	2AF	2AM	2AM	2AM
	Concentration (µg/plate)	2.5	2.5	30	80	2.5	2.50
	Mean no. colonies/plate	1905	124	852	506	2345	489

SA = Sodium azide

2AM = 2-anthramine

9AA = 9-aminoacridine

2NF = 2-nitrofluorene

MMC = Mitomycin C

2AF = 2-aminofluorene

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

 

Table 2: Experiment 2

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type				Frameshift Type
		TA100	TA1535	TA102	WP2uvrA	TA98	TA1537
- - - - - - -	Solvent 50 167 500 1670 5000 10 000	91 83 99 86 94 8 92	10 12 10 9 7 9 8	270 291 252 268 288 272 263	7 6 4 6 6 6 5	29 26 30 28 28 30 29	9 9 8 7 10 11 9
+ + + + + + +	Solvent 50 167 500 1670 5000 10 000	97 101 108 95 94 104 97	8 8 8 4 5 7 5	312 332 302 298 315 318 286	8 5 6 5 7 6 9	31 35 35 32 34 35 34	11 14 11 8 9 11 7
		Positive Controls
-	Name	SA	SA	MMC	ENNG	2NF	9AA
	Concentration (µg/plate)	10	10	2.5	2	5	150
	Mean no. colonies/plate	958	1023	1343	527	523	1746
+	Name	2AM	2AM	2AF	2AM	2AM	2AM
	Concentration (µg/plate)	2.5	2.5	30	80	2.5	2.5
	Mean no. colonies/plate	1533	113	744	508	1821	496

SA = Sodium azide

2AM = 2-anthramine

9AA = 9-aminoacridine

2NF = 2-nitrofluorene

MMC = Mitomycin C

2AF = 2-aminofluorene

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material was considered to be non-mutagenic.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guideline OECD 471 under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at six dose levels, both with and without metabolic activation. The dose levels assessed were 50, 167, 500, 1670, 5000 and 10 000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

The vehicle and positive controls were within the normal range.

The test material was considered to be non-mutagenic under the conditions of this test.