Sorbitan monolaurate, ethoxylated

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(solvent control of positive control substances was not investigated)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(solvent control of positive control substances was not investigated)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sorbitan monolaurate, ethoxylated

EC Number:

500-018-3

EC Name:

Sorbitan monolaurate, ethoxylated

Cas Number:

9005-64-5

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Sorbitan monolaurate, ethoxylated (1-6.5 moles ethoxylated)

Details on test material:

- Name of test material: PC-2012-412
- Molecular formula: UVCB
- Physical state: yellow viscous liquid
- Analytical purity: 100%
- Batch No.: ES61C86614
- Expiration date of the batch: 05 January 2014
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

his and trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Glucose agar medium contained: 18 g/L purified agar in Vogel-Bonner Medium E, 20 g/L glucose. The agar plates also contained 12.5 µg/plate biotin and 15 µg/plate histidine.
Strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Glucose agar medium contained: 18 g/L purified agar in Vogel-Bonner Medium E, 20 g/L glucose. The agar plates also contained 15 µg/plate tryptophan.
The sensitivity of the strain to a wide variety of mutagens was enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 : 10, 33, 100, 333, 1000, 3330 µg/plate in the presence and abscence of 5% S9-mix (all strains); 3 and 5000 µg/plate were additionally tested in TA100 and WP2uvrA (testing 3-5000 µg/plate in tester strains TA100 and WP2uvrA was the dose-range finding test of the study and reported as part of the first experiment)
Experiment 2: 33, 100, 333, 1000, 3330, 5000 µg/plate in the presence and abscence of 5% S9-mix (only tested in TA1535 and TA98 since no biologically relevant toxicity or precipitate on the plates was observed in theses strains in experiment 1)
Experiment 3: 33, 100, 333, 1000, 3330, 5000 µg/plate in the presence and absence of 10% S9-mix (all strains)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (test substance), DMSO and saline (positive controls)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (vehicle of the negative control)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "Details on test system and conditions"

Details on test system and experimental conditions:

Positive control substances:
-S9: sodium azide (5 μg/plate in saline) for TA1535; ICR-191 (2.5 μg/plate in DMSO) for TA1537; 2-nitrofluorene (10 μg/plate in DMSO) for TA98, methylmethanesulfonate (650 μg/plate in DMSO) for TA100, 4-nitroquinoline N-oxide (10 μg/plate in DMSO) for WP2uvrA
+S9 (5%): 2-aminoanthracene in DMSO for all strains (TA1535, TA1537: 2.5 µg/plate; TA98, TA100: 1 µg/plate; WP2uvrA: 10 µg/plate)
+S9 (10%): 2-aminoanthracene in DMSO for all strains (TA1535, TA100: 2.5 µg/plate; TA1537: 5 µg/plate; TA98: 1 µg/plate; WP2uvrA: 10 µg/plate)

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4h at 37°C in the dark

NUMBER OF REPLICATIONS: 3 replications each in 2 (for tester strains TA 1537, TA 100 and WP2 uvrA) or 3 independent experiments (for TA 1535 and TA 98)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test substance was considered negative (not mutagenic) in the test if:
The total number of revertants in tester strain TA100 was not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA was not greater than three times the concurrent vehicle control.
The negative response was reproducible in at least one independently repeated experiment.

A test substance was considered positive (mutagenic) in the test if:
The total number of revertants in tester strain TA100 was greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA was greater than three times the concurrent vehicle control.
In case a repeat experiment was performed when a positive response was observed in one of the tester strains, the positive response was reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "Additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: In a dose range finding test (reported as part of experiment 1), the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 3330 and 5000 μg/plate in the absence of S9-mix and at 1000 µg/plate and above in the presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 2, cytotoxicity was observed in tester strain TA1535 at the test substance concentrations of 3330 and 5000 µg/plate in the absence of S9-mix and at 5000 µg/plate in the presence of S9-mix. In the third experiment, cytotoxic effects were observed in strains TA1535 and TA100 at 3330 and 5000 µg/plate and in TA1537 at 5000 µg/plate without metabolic activation as well as in TA1535 at 5000 µg/plate, in TA1537 at 3330, 5000 µg/plate and in TA100 at 333, 1000, 3330, 5000 µg/plate with metabolic activation.

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): Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain (the S9 -mix contained 5% (v/v) S9 fraction)

Dose (µg/plate)	S9-mix	TA1535	TA1537	TA98	TA100	WP2uvrA
Positive control	-	854 ± 6	811 ± 35	1011 ± 17	879 ± 7	1131 ± 4
Solvent control	-	12 ± 5	5 ± 3	30 ± 8	120 ± 5	22 ± 4
3	-	n.d.	n.d.	n.d.	128 ± 24	30 ± 3
10	-	7 ± 1	4 ± 2	29 ± 6	141 ± 25	19 ± 8
33	-	12 ± 2	5 ± 2	29 ± 5	113 ± 7	21 ± 1
100	-	12 ± 3	3 ± 2	31 ± 2	104 ± 9	29 ± 4
333	-	11 ± 4	6 ± 2	32 ± 5	98 ± 8	28 ± 1
1000	-	9 ± 3	6 ± 2	29 ± 4	64 ± 17	31 ± 7
3330	-	3 ± 1	0 ± 0	34 ± 6	26 ± 2	29 ± 5
5000	-	n.d.	n.d.	n.d.	16 ± 5	30 ± 7
Positive control	+	389 ± 16	387 ± 29	1059 ± 76	1289 ± 33	538 ± 17
Solvent control	+	11 ± 3	4 ± 1	41 ± 9	105 ± 7	22 ± 4
3		n.d.	n.d.	n.d.	104 ± 18	21 ± 2
10	+	9 ± 1	4 ± 1	38 ± 1	109 ± 2	27 ± 3
33	+	10 ± 6	4 ± 1	41 ± 6	103 ± 9	21 ± 1
100	+	12 ± 3	2 ± 2	39 ± 11	96 ± 27	22 ± 3
333	+	9 ± 3	4 ± 2	36 ± 4	128 ± 33	26 ± 4
1000	+	5 ± 3	2 ± 1	35 ± 4	57 ± 6 s	32 ± 8
3330	+	3 ± 1	0 ± 1	41 ± 3	3 ± 2 m	39 ± 6
5000	+	n.d.	n.d.	n.d.	MC m/e	27 ± 6

(testing 3-5000 µg/plate in tester strains TA100 and WP2uvrA represent the dose-range finding test of the study and were reported as part of the first experiment)

solvent control: 0.1 mL ethanol

s: Bacterial background lawn slightly reduced

m: Bacterial background lawn moderately reduced

e: Bacterial background lawn extremely reduced

MC: Microcolonies

Table 2 (Experiment 2): Mean number of revertant colonies/3 replicate plates (± S.D.) with two strains of Salmonella typhimurium (the S9 -mix contained 5% (v/v) S9 fraction)

Dose (µg/plate)	S9-mix	TA1535	TA98
Positive control	-	648 ± 54	845 ± 15
Solvent control	-	9 ± 3	21 ± 5
33	-	6 ± 2	19 ± 1
100	-	8 ± 2	22 ± 3
333	-	8 ± 2	22 ± 3
1000	-	7 ± 1	21 ± 1
3330	-	2 ± 1	20 ± 2
5000	-	1 ± 1	17 ± 3
Positive control	+	218 ± 24	838 ± 39
Solvent control	+	6 ± 2	29 ± 6
33	+	5 ± 1	23 ± 2
100	+	8 ± 2	26 ± 3
333	+	9 ± 0	24 ± 1
1000	+	7 ± 2	23 ± 4
3330	+	4 ± 1	21 ± 3
5000	+	0 ± 0	15 ± 4

(only TA1535 and TA98 were tested in experiment 2, since no biologically relevant toxicity or precipitate on the plates were observed in theses strains in experiment 1)

solvent control: 0.1 mL ethanol

Table 3 (Experiment 3): Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain (the S9 -mix contained 10% (v/v) S9 fraction)

Dose (µg/plate)	S9-mix	TA1535	TA1537	TA98	TA100	WP2uvrA
Positive control	-	792 ± 19	878 ± 44	949 ± 46	813 ± 44	837 ± 105
Solvent control	-	11 ± 2	2 ± 1	15 ± 4	95 ± 9	29 ± 5
33	-	11 ± 3	3 ± 1	21 ± 4	104 ± 7	n.d.
100	-	12 ± 4	5 ± 2	18 ± 3	101 ± 10	26 ±6
333	-	10 ± 1	6 ± 3	22 ± 4	99 ± 8	29 ± 7
1000	-	9 ± 4	4 ± 2	18 ± 2	66 ± 13	29 ± 4
3330	-	2 ± 2	2 ± 2	17 ± 1	23 ± 2	24 ± 2
5000	-	1 ± 1	0 ± 1	17 ± 2	3 ± 3	20 ± 4
Positive control	+	161 ± 23	293 ± 33	1007 ± 123	966 ± 45	300 ± 45
Solvent control	+	6 ± 3	6 ± 3	23 ± 6	69 ± 2	23 ± 5
33	+	6 ± 1	2 ± 1	29 ± 2	71 ± 7	n.d.
100	+	7 ± 3	6 ± 3	26 ± 2	65 ± 2	27 ± 2
333	+	9 ± 1	8 ± 2	28 ± 3	49 ± 15	23 ± 5
1000	+	6 ± 2	2 ± 2	22 ± 3	37 ± 4 s	23 ± 4
3330	+	3 ± 2	0 ± 0	22 ± 4	10 ± 2 m	23 ± 3
5000	+	1 ± 1	0 ± 1	22 ± 2	MC m/e	21 ± 4

solvent control: 0.1 mL ethanol

s: Bacterial background lawn slightly reduced

m: Bacterial background lawn moderately reduced

e: Bacterial background lawn extremely reduced

MC: Microcolonies

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative