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Diss Factsheets

Toxicological information

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
Study period:
17 July - 04 August 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): UY-330
- Physical state: solid
- Appearance: white powder
- Batch No.: CE-201
- Expiration date of the lot/batch: 01 Jan 2005
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
his operon (S. typhimurium); trp operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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:
First experiment: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate with and without metabolic activation (TA 100 and WP2 uvr A; used as range-finding study); 3, 10, 33, 100 and 333 µg/plate with and without metabolic activation (TA 1535, TA 1537 and TA 98)
Second experiment: 3, 10, 33, 100 and 333 µg/plate with and without metabolic activation (all strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO), 0.1 mL/plate
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: sodium azide (5 µg/plate; TA1535), 9-aminoacridine (60 µg/plate; TA1537), daunomycin (4 µg/plate; TA98), methylmethanesulfonate (650 µg/plate; TA100), 4-nitroquinoline N-oxide (10 µg/plate; WP2uvrA); +S9: 2-aminoanthracene (1-5 µg/plate; all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two different experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn
Evaluation criteria:
A test substance is considered mutagenic in the test if it induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. Furthermore, the positive response should be reproducible in at least one independently repeated experiment.
A test substance is considered negative not mutagenic in the test if the total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation. Additionally, the negative response should be reproducible in at least one independently repeated experiment.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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 examined
Positive controls validity:
valid
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the top agar at concentrations of 100 and 333 µg/plate. On the plates, precipitation was observed at 333 µg/plate.
- Metabolic activation: In the first experiment, 5% (v/v) S9 fraction was added to the S9 mix and in the second experiment 10% (v/v) S9 fraction in the S9 mix was used. Therefore, the concentrations of the positive control in the second experiment were twice as high as in the first experiment.
The higher concentration of S9 fraction in the second experiment did not influence the results.

RANGE-FINDING/SCREENING STUDIES: To find an appropriate concentration range for the main study, the test substance was tested in concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the tester strains TA 100 and WP2 uvr A with and without metabolic activation. The range finding study was conducted as a part of experiment 1.
The test substance precipitated in the top agar at concentrations of 100 µg/plate and higher. On the plates, precipitation was observed at concentrations of 333 µg/plate and higher.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values were within the historical control data ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutagenic response of UY-330 in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay – Experiment 1

UY-330: Bacterial Reverse Mutation Assay, mean revertants colonies/plate

EXPERIMENT 1

 

Without S9-Mix

 

Test item (µg/plate)

TA1535

TA 1537

TA 98

TA 100

E.coli

DMSO

9 ± 4

7 ± 1

20 ± 3

138 ± 6

9 ± 2

3

12 ± 3

6 ± 2

18 ± 2

153 ± 12

8 ± 3

10

7 ± 2

7 ± 2

19 ± 3

140 ± 12

7 ± 2

33

12 ± 1

9 ± 1

16 ± 2

150 ± 12

9 ± 2

100

12 ± 3

12 ± 2

21 ± 2

148 ± 8

10 ± 1

333

14 ± 1 P

7 ± 3 P

16 ± 5 P

134 ± 9 P

9 ± 3 P

1000

---

---

---

132 ± 10 P

9 ± 3 P

3330

---

---

---

120 ± 9 P

9 ± 3 P

5000

---

---

---

124 ± 6 P

9 ± 2 P

sodium azide

354 ± 42

---

---

---

---

9-aminoacridine

---

241 ± 65

---

---

---

daunomycin

---

---

541 ± 62

---

---

MMS

---

---

---

962 ± 22

---

4-NQO

---

---

---

---

812 ± 73

 

With S9-Mix (5% v/v S9 fraction*)

 

Test item (µg/plate)

TA1535

TA 1537

TA 98

TA 100

E.coli

DMSO

14 ± 3

10 ± 3

25 ± 7

140 ± 21

10 ± 1

3

11 ± 3

5 ± 2

17 ± 2

134 ± 23

13 ± 3

10

13 ± 3

8 ± 3

23 ± 3

148 ± 15

10 ± 1

33

12 ± 2

7 ± 3

26 ± 4

139 ± 9

10 ± 1

100

11 ± 2

7 ± 1

25 ± 6

146 ± 15

14 ± 3

333

11 ± 2 P

10 ± 2 P

22 ± 1 P

138 ± 12 P

11 ± 3 P

1000

---

---

---

131 ± 13 P

10 ± 1 P

3330

---

---

---

130 ± 12 P

8 ± 3 P

5000

---

---

---

131 ± 22 P

8 ± 1 P

2-AA

167 ± 16

229 ± 28

741 ± 26

717 ± 50

73 ± 11

DMSO: dimethyl sulfoxide

MMS: methylmethane sulfonate

4-NQO: 4-nitroquinoline N-oxide

2-AA: 2-aminoanthracene

P: precipitation

*Before use, all S9 batches were characterized with 5 µg/plate benzo[a]pyrene, which requires metabolic activation, in the tester strain TA 98.

Table 2: Mutagenic response of UY-330 in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay – Experiment 2

UY-330: Bacterial Reverse Mutation Assay, mean revertants colonies/plate

EXPERIMENT 2

 

Without S9-Mix

 

Test item (µg/plate)

TA1535

TA 1537

TA 98

TA 100

E.coli

DMSO

9 ± 2

8 ± 2

12 ± 3

132 ± 8

7 ± 2

3

11 ± 3

7 ± 2

16 ± 1

137 ± 15

8 ± 2

10

11 ± 5

7 ± 1

18 ± 3

141 ± 10

11 ± 5

33

13 ± 6

6 ± 1

13 ± 4

156 ± 3

13 ± 1

100

10 ± 3

6 ± 2

12 ± 3

130 ± 8

8 ± 1

333

13 ± 4 P

10 ± 6 P

15 ± 3 P

136 ± 8 P

9 ± 1 P

sodium azide

355 ± 75

---

---

---

---

9-aminoacridine

---

297 ± 6

---

---

---

daunomycin

---

---

326 ± 57

---

---

MMS

---

---

---

965 ± 32

---

4-NQO

---

---

---

---

1323 ± 72

 

With S9-Mix (10% v/v S9 fraction*)

 

Test item (µg/plate)

TA1535

TA 1537

TA 98

TA 100

E.coli

DMSO

11 ± 4

10 ± 4

19 ± 6

135 ± 9

8 ± 3

3

10 ± 7

7 ± 2

17 ± 6

137 ± 14

10 ± 2

10

7 ± 4

9 ± 1

16 ± 7

136 ± 14

9 ± 2

33

9 ± 5

10 ± 2

17 ± 8

147 ± 3

9 ± 2

100

7 ± 5

8 ± 2

17 ± 6

150 ± 11

9 ± 3

333

10 ± 6 P

7 ± 3 P

18 ± 8 P

136 ± 9 P

9 ± 1 P

2-AA

136 ± 6

218 ± 31

196 ± 35

829 ± 14

120 ± 13

DMSO: dimethyl sulfoxide

MMS: methylmethane sulfonate

4-NQO: 4-nitroquinoline N-oxide

2-AA: 2-aminoanthracene

P: precipitation

*Before use, all S9 batches were characterized with 5 µg/plate benzo[a]pyrene, which requires metabolic activation, in the tester strain TA 98.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative