Fatty acids C16-18 (even numbered), mono and di esters with Sucrose

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 - 25 April

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

missing 5th strain (TA 102 or E. coli WP2)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

yes

Remarks:

missing 5th strain (TA 102 or E. coli WP2)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

Sep 1985

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids C16-18 (even numbered), mono and di esters with Sucrose

EC Number:

947-854-9

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Fatty acids C16-18 (even numbered), mono and di esters with Sucrose

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Test substance is well soluble in DMSO at approx. 48 °C and is stabil for 4 h.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in 2 different experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of colonies or a thinning of the bacterial background lawn

Evaluation criteria:

A test compound is classified as mutagenic if it has either of the following effects:
a) a test compound produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of
revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test compound induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of
revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
lf the test substance does not produce reproducible increases of at least 2 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this system.
The test results must be reproducible.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1: +S9: at and above 2500 µg/plate; -S9: at and above 500 µg/plate Exp 2: +S9: at and above 500 µg/plate; -S9: at and above 100 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1: +S9: at and above 2500 µg/plate; -S9: at and above 100 µg/plate Exp 2: +/- S9: at and above 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1 and 2: +S9: at and above 2500 µg/plate; -S9: at and above 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1: +S9: at and above 5000 µg/plate; -S9: at and above 500 µg/plate Exp 2: +/- S9: at and above 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Visible precipitation of the test substance was observed at 2500 µg/plate and above in both experiments.

RANGE-FINDING/SCREENING STUDIES: In a toxicity test with a dilution of tester strain TA 100, which was performed in parallel with the second experiment, toxicity was found at a concentration of 2500 μg/plate in the presence of metabolic activation and 100 μg/plate in the absence of metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both mutagenicity experiments strain dependent cytotoxicity was observed in a dose range of 100 to 2500 μg/plate and above without metabolic activation and in a dose range of 500 to 5000 μg/plate withmetabolic activation. Thinning of bacterial
lawn and in most cases also a reduction in the number of colonies were observed at these doses.

Any other information on results incl. tables

Table 1: Results of Experiment 1

	Number of revertant colonies (mean of 3 plates±SD)
With S9
Test substance (µg/plate)	TA 1535	TA 1537			TA 98	TA 100
Solvent control	10.7±1.2	14.7±1.2			26.7±5.5	115.3±5.0
Negative control (untreated)	10.3±1.5	17.0±4.6			22.3±1.5	148.3±4.0
0	10.7±1.2	14.7±1.2			26.7±5.5	115.3±5.0
4	13.3±1.5	14.3±2.5			26.7±4.7	123.7±6.7
20	11.7±3.5	14.3±1.5			21.0±1.7	132.7±8.0
100	9.3±2.3	13.0± 1.0			22.7±2.1	126.0±2.6
500	9.7±0.6	14.7±3.5			19.7±2.5	121.7±3.2
2500	7.0±1.7 r	2.7±1.2 r			16.3±6.7 r	93.3±10.1
500	3.0±2.0 r	1.0±0.0 c			8.3±2.5 r	102.0±15.6 r
Positive Control	2-AA	2-AA			2-AA	2-AA
Dose (µg/plate)	1.0 µg/plate	1.0 µg/plate			0.5 µg/plate	0.5 µg/plate
Number of revertant colonies/plate	194.3±9.3	288.7±33.2			2211.7±0.5	1757.7±69.6
Without S9
Test substance (µg/plate)	TA 1535		TA 1537		TA 98	TA 100
Solvent control	8.3±0.6		12.0±2.0		19.7±3.2	116.3±15.3
Negative control (untreated)	11.7±0.6		12.7±1.2		25.0±3.6	119.3±15.2
0	8.3±0.6		12.0±2.0		19.7±3.2	116.3±15.3
4	12.7±0.6		8.3±3.1		22.7±2.9	107.7±11.9
20	10.3±2.9		8.0±3.0		19.3±2.5	129.0±17.6
100	9.7±2.1		5.0±3.6 r		21.3±2.9	117.0±5.3
500	10.0±2.0 r		5.0±1.7 r		12.7±2.1 r	83.7±7.6
2500	1.7±0.6 r		2.7±1.2c		6.3±0.6 r	93.0±9.5
5000	1.3±0.6 r		1.0±0.0 c		6.7± 3.1 c	83.3±14.4 r
Positive Control	NaN3		9-AA		2-NF	NaN3
Dose (µg/plate)	2.5		2.5		2.5	2.5
Number of revertant colonies/plate	461.3±2.3		150.7±43.1		564.3±67.5	636.3±10.1
2AA: 2-Aminoanthracene 9-AA: 9-Aminoacridine 2-NF: 2-Nitrofluorene NaN3: sodium azide				r: reduced background growth c: clearing of background growth

Table 2. Results of Experiment 2

	Number of revertant colonies (mean of 3 plates±SD)
With S9
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100
Solvent control	12.3±1.2	9.7±2.5	31.3±5.1	130.3±0.6
Negative control (untreated)	12.3±1.5	10.3±0.6	26.0±2.6	163.3±13.3
0	12.3±1.2	9.7±2.5	31.3±5.1	130.3±0.6
4	9.3±7.6	9.0±1.0	32.7±6.5	137.7±12.4
20	8.3±5.5	8.3±1.2	23.7±2.1	139.0±11.5
100	10.7±3.1	10.3±3.2	28.0±3.0	149.3±5.1
500	12.3±2.9 r	4.3±2.5 r	30.3±12.1	143.7±10.1
2500	6.3±2.9 r	3.0±1.7 r	12.3±2.1 r	129.7±0.6 r
500	6.3± 6.7 r	2.0±1.0 c	10.3±1.2 r	135.3±14.2 r
Positive Control	2-AA	2-AA	2-AA	2-AA
Dose (µg/plate)	1.0 µg/plate	1.0 µg/plate	0.5 µg/plate	0.5 µg/plate
Number of revertant colonies/plate	172.7±13.1	272.3±13.8	2199.7±150.5	1897.7±114.2
Without S9
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100
Solvent control	13.7±0.6	8.0±0.0	20.3±1.2	130.3±0.6
Negative control (untreated)	13.7±2.1	7.3±1.2	26.0±4.4	163.3±13.3
0	13.7±0.6	8.0±0.0	20.3±1.2	125.3±15.9
4	11.0±2.6	7.0±1.0	24.0±1.7	129.3±7.0
20	9.0±1.7	8.3±1.2	24.3±1.2	124.0±8.2
100	11.3±3.1 r	7.0±1.0	26.7±3.8	123.7±23.6
500	11.3±2.5 r	2.3±0.6 r	13.0±2.6 r	105.3±19.5
2500	1.7±1.5 r	1.3±0.6 c	6.7±0.6 r	95.0±3.6 r
5000	0.3±0.6 r	1.0±0.0 c	5.0±1.0 r	83.0±7.9 r
Positive Control	NaN3	9-AA	2-NF	NaN3
Dose (µg/plate)	2.5	2.5	2.5	2.5
Number of revertant colonies/plate	431.3±55.0	215.3±33.1	520.3±89.2	1897.7±114.2
2AA: 2-Aminoanthracene 9-AA: 9-Aminoacridine 2-NF: 2-Nitrofluorene NaN3: sodium azide			r: reduced background growth c: clearing of background growth

Applicant's summary and conclusion

Conclusions:

Under the test conditions used, the substance was not mutagenic in any of the four strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) tested with and without metabolic activation.