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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:
2002-05-10 to 2002-09-09
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)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
See table 1
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 (without activation)
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 (without activation)
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without activation)
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102 (without activation)
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthracene amide
Remarks:
TA 98, TA 102, TA 1537 (with activation)
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
TA 100, TA 1535 (with activation)
Details on test system and 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: Relative cell count / background lawn

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.

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 results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: No mutagenic potential
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)

 

TA100

Conc.
(µg/plate)

Plate 1

Plate 2

Cytotoxic
(yes/no)

0*

143

146

No

0.316

154

138

No

1

188

140

No

3.16

171

144

No

10

161

162

No

31.6

144

145

No

100

145

138

No

316

115

130

No

1000

156

135

No

3160

195

154

No

5000

171

146

No

*solvent control with DMSO

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

 

TA98

TA100

TA102

Conc.
µg/plate

— MA

+

MA

Cytotoxic
(yes/no)

MA

+

 MA

Cytotoxic
(yes/no)

 MA

+

MA

Cytotoxic
(yes/no)

0*

28

37

No

150

147.3

No

285.3

282.3

No

100

26.7

35.7

No

129

154.7

No

267

268

No

316

32

40.3

No

141

153.7

No

283.7

264.7

No

1000

29.3

29

No

140.7

147

No

287.3

259.3

No

3160

27.3

39

No

138

135.3

No

274.7

272

No

5000

27.3

36

No

128

150.7

No

263.7

296.7

No

Positive control

575.3

641.7

No

1009.7

941.7

No

1035

1038

No

*solvent control with DMSO

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

 

TA1535

TA1537

Conc.
µg/plate

— MA

+

MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

17.3

18.3

No

6.7

4

No

100

17.3

14.7

No

4.7

6.3

No

316

20.7

18.7

No

6.7

6.3

No

1000

18

13.7

No

5.7

4.7

No

3160

17

18.7

No

8

5.7

No

5000

16.3

17.7

No

6.3

4.7

No

Positive control

740.7

645.3

No

324.3

320

No

*solvent control with DMSO

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

 

TA98

TA100

TA102

Conc.
µg/plate

— MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

 MA

+

MA

Cytotoxic
(yes/no)

0*

42.3

41.7

No

152.3

123.7

No

278.3

279.3

No

100

36.3

31

No

129.7

122

No

282.7

290.3

No

316

30

30

No

128.3

158

No

262.7

270.7

No

1000

32

40

No

117

128.3

No

270.7

272.3

No

3160

23.3

29.3

No

121

142.7

No

274.3

270.7

No

5000

33.3

37.7

No

140.7

117

No

281.3

269

No

Positive control

546.7

533.3

No

1143.7

1139.0

No

1249.7

1249.7

No

*solvent control with DMSO

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

 

TA1535

TA1537

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+

 MA

Cytotoxic
(yes/no)

0*

15.3

14.7

No

4

4.3

No

100

13.3

12.3

No

3.3

3

No

316

12.7

13.7

No

5.0

4.3

No

1000

12

15.7

No

3.3

4.3

No

3160

13

15.7

No

3

3

No

5000

13.7

16

No

4.3

4.3

No

Positive control

321.3

333

No

338.3

348.7

No

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:
In a reliable test conducted in compliance with OECD 471, under 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.