Fatty acids C8-18 (even numbered), mono- and diesters 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:

24 Apr - 31 May 1996

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:

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 in 1983

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

only 4 tester strains used

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids C8-18 (even numbered), mono- and diesters with sucrose

EC Number:

947-862-2

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Fatty acids C8-18 (even numbered), mono- and diesters with sucrose

Method

Target gene:

Histidin 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:

S9 mix (rat liver supernatant from rats treated with Aroclor 1254)

Test concentrations with justification for top dose:

Without and without metabolic activation: 4, 20, 100, 500, 2500, 5000 μg/plate in both experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
The test compound did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate.

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)

SELECTION AGENT (mutation assays): histidine (in agar)

NUMBER OF REPLICATIONS:
1st experiment: Triplicates (3 plates per dose)
2nd experiment: Triplicates (3 plates per dose)

DETERMINATION OF CYTOTOXICITY
A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.

Parallel to the 2nd experiment, precise toxicity testing was performed: 0.1 mL of the different concentrations of the test compound were thoroughly mixed with 0.1 mL of 1.0E+07 dilution of the overnight culture of TA 100 (designated TA 100D) and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control was compared with the number of colonies per plate in the presence of the test compound. Results were given as a ratio of these values (= surviving fraction).

- OTHER:
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A..
The different bacterial strains were checked half-yearly with regard to their respective biotin, histidine requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the toxicity test using histidine enriched agar plates and a dilution of the tester strain TA 100 (designated TA 100 D), which was performed in parallel with the second experiment, toxicity was found at concentrations of 2500 μg/plate and above in the absence of metabolic activation and 5000 μg/plate in the presence of metabolic activation.

Any other information on results incl. tables

Table 1: Results of first experiment (number of revertants)

		plate 1	plate 2	plate 3	Bacterial lawn
TA 100	Solvent control	137	145	146
with S9	Negative control	145	170	160
	4 µg/plate	143	151	147
	20 µg/plate	151	145	99
	100 µg/plate	136	153	145
	500 µg/plate	136	138	145
	2500 µg/plate	124	140	130
	5000 µg/plate	153	162	139	incomplete
	2-aminoanthracene (0.5 µg/plate)	942	914	1052
TA 100	Solvent control	148	133	129
without S9	Negative control	170	165	146
	4 µg/plate	129	116	146
	20 µg/plate	145	142	119
	100 µg/plate	118	134	145
	500 µg/plate	134	147	144
	2500 µg/plate	110	126	110
	5000 µg/plate	111	76	84
	sodium azide (1.0 µg/plate)	634	718	652	incomplete
TA 1535	Solvent control	17	15	20
with S9	Negative control	9	14	12
	4 µg/plate	11	10	16
	20 µg/plate	17	11	11
	100 µg/plate	12	11	11
	500 µg/plate	16	15	12
	2500 µg/plate	10	7	13	incomplete
	5000 µg/plate	8	11	7	incomplete
	2-aminoanthracene (1.0 µg/plate)	129	125	143
TA 1535	Solvent control	10	13	11
without S9	Negative control	9	12	12
	4 µg/plate	14	12	12
	20 µg/plate	10	11	14
	100 µg/plate	10	10	10
	500 µg/plate	11	8	14
	2500 µg/plate	6	8	10	incomplete
	5000 µg/plate	3	4	5	incomplete
	sodium azide (1.0 µg/plate)	385	545	415
TA 1537	Solvent control	10	7	11
with S9	Negative control	8	9	12
	4 µg/plate	9	9	8
	20 µg/plate	7	10	10
	100 µg/plate	7	6	8
	500 µg/plate	8	8	6	incomplete
	2500 µg/plate	6	2	5	incomplete
	5000 µg/plate	3	1	2	incomplete
	2-aminoanthracene (1.0 µg/plate)	125	137	184
TA 1537	Solvent control	6	7	7
without S9	Negative control	9	10	7
	4 µg/plate	6	9	8
	20 µg/plate	8	6	9
	100 µg/plate	6	9	7
	500 µg/plate	4	8	10
	2500 µg/plate	8	3	8	incomplete
	5000 µg/plate	8	4	4	incomplete
	9-aminoacridine (50 µg/plate)	236	117	118
TA98	Solvent control	21	28	21
with S9	Negative control	28	29	29
	4 µg/plate	25	33	30
	20 µg/plate	27	31	30
	100 µg/plate	32	25	32
	500 µg/plate	29	32	28
	2500 µg/plate	22	24	34	incomplete
	5000 µg/plate	28	13	13	incomplete
	2-aminoanthracene (0.5 µg/plate)	1212	1213	1292
TA98	Solvent control	26	21	22
without S9	Negative control	27	23	24
	4 µg/plate	21	19	21
	20 µg/plate	28	22	18
	100 µg/plate	18	17	24
	500 µg/plate	24	22	19
	2500 µg/plate	18	9	18	incomplete
	5000 µg/plate	7	5	7	incomplete
	2-nitrofluorene (2.5 µg/plate)	220	336	265

Table 2: Results of second experiment (number of revertants)

		plate 1	plate 2	plate 3	Bacterial lawn
TA 100	Solvent control	141	150	144
with S9	Negative control	134	144	145
	4 µg/plate	140	123	133
	20 µg/plate	123	139	130
	100 µg/plate	121	150	145
	500 µg/plate	137	158	155
	2500 µg/plate	130	130	120
	5000 µg/plate	119	143	155
	2-aminoanthracene (0.5 µg/plate)	1550	1514	1562
TA 100	Solvent control	127	122	138
without S9	Negative control	173	162	141
	4 µg/plate	117	139	146
	20 µg/plate	135	136	132
	100 µg/plate	124	130	141
	500 µg/plate	116	130	120
	2500 µg/plate	110	112	117
	5000 µg/plate	87	80	76
	sodium azide (1.0 µg/plate)	682	704	685
TA 1535	Solvent control	15	9	10
with S9	Negative control	11	14	15
	4 µg/plate	14	12	9
	20 µg/plate	11	14	11
	100 µg/plate	8	10	12
	500 µg/plate	11	14	12
	2500 µg/plate	14	12	16
	5000 µg/plate	12	10	9
	2-aminoanthracene (1.0 µg/plate)	160	145	193
TA 1535	Solvent control	14	14	10
without S9	Negative control	14	8	8
	4 µg/plate	9	10	10
	20 µg/plate	9	15	11
	100 µg/plate	11	14	12
	500 µg/plate	11	14	14
	2500 µg/plate	12	14	9
	5000 µg/plate	3	2	4	incomplete
	sodium azide (1.0 µg/plate)	464	426	424
TA 1537	Solvent control	7	11	10
with S9	Negative control	7	9	8
	4 µg/plate	8	7	10
	20 µg/plate	10	11	8
	100 µg/plate	11	6	6
	500 µg/plate	8	7	8
	2500 µg/plate	10	10	8
	5000 µg/plate	5	3	3	incomplete
	2-aminoanthracene (1.0 µg/plate)	207	253	256
TA 1537	Solvent control	8	8	6
without S9	Negative control	10	6	9
	4 µg/plate	10	6	10
	20 µg/plate	8	9	6
	100 µg/plate	9	8	14
	500 µg/plate	7	10	9
	2500 µg/plate	4	6	9	incomplete
	5000 µg/plate	3	2	1	incomplete
	9-aminoacridine (50 µg/plate)	149	16	139
TA98	Solvent control	44	31	33
with S9	Negative control	45	26	38
	4 µg/plate	22	29	30
	20 µg/plate	29	31	35
	100 µg/plate	30	24	38
	500 µg/plate	37	30	41
	2500 µg/plate	20	23	30
	5000 µg/plate	14	20	19	incomplete
	2-aminoanthracene (0.5 µg/plate)	2007	1917	1933
TA98	Solvent control	24	27	27
without S9	Negative control	22	23	26
	4 µg/plate	28	21	34
	20 µg/plate	36	34	34
	100 µg/plate	26	28	25
	500 µg/plate	32	33	24
	2500 µg/plate	19	11	16	incomplete
	5000 µg/plate	5	6	8	incomplete
	2-nitrofluorene (2.5 µg/plate)	777	756	765

Applicant's summary and conclusion

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.