Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002-05-10 to 2002-09-9
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:
solid

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:
Aroclor induced rat liver S9-mix
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 0.316, 1, 3.16, 10, 31.6, 100, 316, 1000, 3160 and 5000 μg/plate (without metabolic activation)
Experiment I:
- 100, 316, 1000, 3160 and 5000 μg/plate (with and without metabolic activation)
Experiment II:
- 31.6, 100, 316, 1000 and 3160 μg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethylene glycol dimethylether
- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9-mix: sodium azide, 10 µg/plate (TA 100, TA 1535); 2-nitro-fluorene, 10 µg/plate (TA 98); 9-AA, 100 µg/plate (TA 1537); MMS, 1300 µg/plate (TA 102); +S9-mix: 2-AA, 2 µg/plate (TA 98, TA 102, TA 1537); CPA, 1500 µg/plate (TA 100, TA 1535)
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 assessment, 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
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3160 μg/plate for all tester strains (experiment II with and without S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3160 μg/plate (experiment II with and without S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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)

 

TA 100

Conc.
(µg/plate)

Plate 1

Plate 2

Cytotoxic
(yes/no)

0*

123

131

No

0.316

113

118

No

1

117

106

No

3.16

108

179

No

10

108

116

No

31.6

105

129

No

100

139

149

No

316

140

149

No

1000

130

124

No

3160

159

145

No

5000

192

189

No

*solvent control with Ethylene glycol dimethylether

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

 

TA 98

TA 100

TA 102

Conc.
µg/plate

 MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

 MA

+

 MA

Cytotoxic
(yes/no)

0*

29.7

28

No

128.3

122.7

No

297.3

297.3

No

100

26.7

24.7

No

130.7

144.7

No

308

274.7

No

316

32

37.3

No

131.7

129.3

No

302

264.7

No

1000

39.3

31.7

No

130.3

142

No

298.7

299

No

3160

34.3

33.7

No

123.3

128

No

290.3

276

No

5000

40

30

No

126

125.7

No

294

273.7

No

Positive control

916

944.3

No

1022.3

1007

No

1051

1027.7

No

*solvent control with Ethylene glycol dimethylether

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

 

TA 1535

TA 1537

Conc.
µg/plate

MA

+

 MA

Cytotoxic
(yes/no)

MA

+

 MA

Cytotoxic
(yes/no)

0*

14.3

21.3

No

7

4.7

No

100

14

15.7

No

5

3.3

No

316

15.3

14.7

No

5.7

5.7

No

1000

18.7

14

No

4.3

3.7

No

3160

15.7

14

No

5

7.7

No

5000

18.3

12.7

No

3.7

5.3

No

Positive control

404.7

410.7

No

407.3

414.3

No

*solvent control with Ethylene glycol dimethylether

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

 

TA 98

TA 100

TA 102

Conc.
µg/plate

MA

+

 MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+

 MA

Cytotoxic
(yes/no)

0*

34

36.3

No

126.7

147

No

277

280.7

No

31.6

31

38.7

No

138.3

109

No

284

275

No

100

28

29.7

No

126

121.7

No

287.3

269

No

316

31.7

36.3

No

140.7

133.3

No

266

279

No

1000

33.3

32.7

No

132.7

134.7

No

271.3

272.3

No

3160

0

0

Yes

0

0

Yes

0

0

Yes

Positive control

538.7

557.7

No

1122

1195.3

No

1335.3

1240.7

No

*solvent control with Ethylene glycol dimethylether

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

 

TA 1535

TA 1537

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

12.7

14

No

5.7

4.3

No

31.6

13.3

14

No

4

4.3

No

100

14

12.3

No

4

5

No

316

11.7

14

No

5

5

No

1000

13.7

12

No

5.7

6.7

No

3160

0

0

Yes

0

0

Yes

Positive control

337

333

No

336.3

339

No

*solvent control with Ethylene glycol dimethylether

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.