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Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(solvent control of positive control substances was not investigated)
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

Constituent 1
Chemical structure
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
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 / strainopen allclose all
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 / 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