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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 June 2002 to 02 December 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Freie und Hansestadt Hamburg; Behörde für Arbeit, Gesundheit und Soziales
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): VP 50991
- Lot/batch No.: QB 14002
- Expiration date of the lot/batch: 2004-01-01
- Stability under test conditions: at 4°C in the dark: >1 day
- Storage condition of test material: at room temperature, dark, tightly closed, dry

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
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:
See table 1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide: TA100, TA1535, 10 µg/plate; 2-nitrofluorene: TA98, 10 µg/plate; 9-aminoacridine: TA1537, 100 µg/plate; methylmethanesulfonate: TA102, 1300 µg/plate; without metabolic activation
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthracene amide: TA 98, TA 102, TA 1537, 2 µg/plate; cyclophosphamide: TA 100, TA 1535; 1500 µg/plate
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION

- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn evaluation, revertant colony counts
Evaluation criteria:
A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both experiments.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking on histidine-free agar plates.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.
Statistics:
MANN and WHITNEY and Spearman’s rank.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 - 1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
316 - 1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 - 1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
316 - 1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 - 1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
316 - 1000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Any other information on results incl. tables

Table 2: Dose range-finding study Number of revertants per plate (2 plates)

 Strain

TA100

 Background lawn

Conc.
(µg/plate)

Plate 1

normal

0*

145

normal

0.316

157

normal

1

155

normal

3.16

173

normal

10

149

normal

31.6

152

normal

100

169

normal

316

179

normal

1000

177

scarce

3160

0

scarce

5000

0

scarce

*solvent control with DMSO

 

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

 Strain

TA98

TA100

TA102

Conc.
µg/plate

- 

MA

+

MA

Cytotoxic
(yes/no)

- 

MA

+

MA

Cytotoxic
(yes/no)

-

 MA

+

MA

Cytotoxic
(yes/no)

0*

20.3

35.3

No

118.3

143

No

288.7

299.3

No

10

22.3

39.3

No

140.7

139.7

No

268.3

274

No

31.6

22.3

39

No

104.3

180.7

No

277.3

278.3

No

100

20.7

41.3

No

112.7

133.3

No

200**

159

Yes

316

27.7

27

No

122.3

146

No

207.7**

113.3**

Yes

1000

0**

5.7**

Yes

96.3**

110.7**

Yes

154**

0**

Yes

Positive control

755.3

758.7

No

1123

413.3

No

1346

1061

No

*solvent control with DMSO

** scarce background lawn

 

Table 3: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates)

Strain

TA1535

 

 

TA1537

Conc.
µg/plate

-

MA

+

MA

Cytotoxic
(yes/no)

-

MA

+

MA

Cytotoxic
(yes/no)

0*

9

14.3

No

8.3

15

No

10

11

10

No

8.3

8

No

31.6

6.7

12

No

10.7

6.3

No

100

9.7

14.7

No

9.0**

8

No

316

15.3**

16

Yes

4.3**

9

Yes

1000

0**

5.3**

Yes

1**

0**

Yes

Positive control

291.7

240

No

246.7

254.3

No

*solvent control with DMSO

** scarce background lawn

 

 

Table 4: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

 Strain

TA98

TA100

TA102

Conc.
µg/plate

- 

MA

+ MA

Cytotoxic
(yes/no)

- 

MA

+ MA

Cytotoxic
(yes/no)

- 

MA

+ MA

Cytotoxic
(yes/no)

0*

24

49

No

110.3

102.3

No

259

336

No

3.16

24.3

40

No

94.7

90.3

No

270.3

331.3

No

10

27

40.3

No

103

104.3

No

258.7

343.3

No

31.6

22.3

38.3

No

96.7

118.3

No

228

273.3

No

100

14**

37

Yes

97.3

104.7

Yes

90.7**

272.3

Yes

316

5**

28.3

Yes

42**

106

Yes

0**

0**

Yes

Positive control

683.7

676

No

1065.3

1092

No

1076.3

1101

No

*solvent control with DMSO

** scarce background lawn

 

 

Table 5: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

Strain

TA1535

TA1537

Conc.
µg/plate

-MA

+ MA

Cytotoxic
(yes/no)

- 

MA

+

MA

Cytotoxic
(yes/no)

0*

12.3

12.7

No

9

11.3

No

3.16

20

19.7

No

8.7

8.7

No

10

19

14.3

No

5.7

6

No

31.6

15.7

11.7

No

5.7

9.3

No

100

18.7**

14.3

Yes

5

8.3

No

316

0**

12**

Yes

1**

0.3**

Yes

Positive control

237

318

No

287.3

283.3

No

*solvent control with DMSO

** scarce background lawn

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

In a highly reliable test, conducted under OECD 471, with GLP, no mutagenic effect was observed for the test substance tested up to cytotoxic concentration in any of the test strains in two independent experiments without and with metabolic activation. The test substance is non-mutagenic in test strains used.