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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:
09 April 2002 to 01 July 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)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
FREIE UND HANSESTADT HAMBURG, BEHÖRDE FÜR ARBEIT, GESUNDHEIT UND SOZIALES, Hamburg (Germany)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): BS 1316 (Test substance No. 60006325)
- Physical state: colourless liqiud at 20 °C
- Lot/batch No.: KE 01282
- Expiration date of the lot/batch: 01 Jan 2004
- Stability under test conditions: at least 1 week at +4°C in the dark (in solvent)
- Storage condition of test material: at room temperature, dark
- Density: 0.90 g/cm³
- Melting point: < -78 °C
- pH value: approx. 7
- Receipt No.: 24388
- Date of receipt: 21 Feb 2002

Method

Target gene:
his-operon
Species / strain
Species / strain / cell type:
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:
- Test 1: 100, 316, 1000, 3160 and 5000 µg/plate (plate incorporation test)
- Test 2: 3.16, 10, 31.6, 100 and 316 µg/plate (preincubation test)
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:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
methylmethanesulfonate
other: +S9: 2-anthracene amide (TA 98, TA 102, TA 1537; 2µg/plate)
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 in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn assessment, revertant colony counts (only TA 100)

ACTIVATION: Aroclor induced rat liver S9 had a protein content of 32.92 mg/ml. the S9 mix included 5% S9 and glucose-6-phosphate and NADP as co-factors. 0.5 ml S9 mix was added to 2 ml top agar, 0.1 ml of cell suspension and 0.1 ml test material (or solvent or positive control), giving a final concentration of approximately 1% S9
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 results
Species / strain:
S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 100 µg/plate in TA 100 ±S9, and 316 µg/plate in TA 98 and TA 100 ±S9 and in TA 1535 and TA 1537 -S9.
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 1: Dose range-finding study: number of revertants per plate (2 plates)

 

TA 100

Conc.
(µg/plate)

Plate 1

Plate 2

Cytotoxic
(yes/no)

0*

132

175

No

0.316

150

195

No

1

127

125

No

3.16

169

152

No

10

138

135

No

31.6

164

149

No

100

128

133

No

316

154

126

No

1000

133

174

No

3160

151

140

No

5000

163

167

Yes

*solvent control with Ethylene glycol dimethylether

Table 2: 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*

26.7±7.6

30.3±4.5

No

123.3±10.1

167.7±7.4

No

302.0±8.7

287.3±14.8

No

100

24.0±2.6

47.7±5.5

No

151.7±19.7

147.3±8.0

No

240.3±17.6

245.7±9.9

No

316

30.7±8.5

34.7±2.1

No

150.7±9.3

151.0±2.6

No

234.7±6.4

225.0±19.1

No

1000

20.3±3.8

34.0±3.0

No

159.3±1.2

144.7±3.2

No

226.3±29.9

245.7±17.9

No

3160

26.0±7.0

35.3±4.7

No

204.0±7.2

158.7±4.5

No

248.7±35.6

224.0±22.3

No

5000

24.0±6.2

28.7±3.2

No

192.7±15.9

173.3±10.4

Yes

259.3±23.9

206.3±20.6

Yes

Positive control

331.3±23.7

343.7±2.5

No

1091.3±37.8

1061.3±14.4

No

1087.7±12.0

1159.3±70.9

No

*solvent control with ethylene glycol dimethylether

Table 2: 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*

19.0±4.4

16.3±1.5

No

9.3±1.5

11.7±0.6

No

100

17.7±5.9

14.3±4.0

No

9.7±2.1

11.7±3.8

No

316

19.0±7.0

15.0±4.6

No

7.7±3.1

12.0±2.0

No

1000

17.3±3.2

17.3±2.3

No

11.0±1.0

13.7±1.5

No

3160

17.7±0.6

19.3±5.7

No

9.0±2.0

15.0±2.6

No

5000

19.0±1.7

15.3±6.7

No

11.3±2.5

14.0±3.6

No

Positive control

387.0±7.2

393.7±1.2

No

1000.0±5.2

986.0±3.5

No

*solvent control with ethylene glycol dimethylether

Table 3: 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*

26.7±9.0

34.7±2.5

No

125.0±14.8

119.3±8.7

No

291.0±7.5

280.3±13.3

No

3.16

31.3±2.1

31.7±11.4

No

133.0±12.1

117.3±15.9

No

291.3±1.5

267.7±27.4

No

10

28.7±2.5

30.7±5.5

No

186.7±3.1

127.3±8.5

No

263.7±11.2

260.7±7.4

No

31.6

39.3±16.2

31.7±4.5

No

155.7±8.1

106.0±9.6

No

265.3±0.6

278.0±28.6

No

100

28.3±5.0

31.0±4.0

No

144.3±15.5

124.0±2.6

No

0.0±0.0

270.3±16.3

Yes

316

33.7±3.5

30.0±1.0

Yes

113.7±7.5

112.7±8.4

Yes

0.0±0.0

0.0±0.0

Yes

Positive control

855.7±22.7

924.0±54.7

No

1003.0±7.5

926.0±119.7

No

1165.3±6.1

1200.7±77.6

No

*solvent control with ethylene glycol dimethylether

Table 3: 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*

12.0±1.0

14.7±2.1

No

5.7±3.8

10.0±1.0

No

3.16

14.0±3.6

15.0±1.7

No

6.7±2.5

6.7±1.5

No

10

14.7±0.6

12.3±4.2

No

7.3±3.2

10.3±1.5

No

31.6

14.0±4.0

15.0±2.6

No

5.3±1.2

9.3±2.1

No

100

12.3±1.5

12.7±2.1

No

6.3±1.5

7.7±1.5

No

316

16.0±1.0

0.0±0.0

Yes

10.3±1.5

0.0±0.0

Yes

Positive control

632.0±6.1

581.7±46.3

No

617.0±4.6

423.3±86.4

No

*solvent control with ethylene glycol dimethylether

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without metabolic activation

The test item has been tested in a reliable study conducted according to the OECD TG 471 and in compliance with GLP. No test-substance related increase in the number of revertants was observed when tested with and without metabolic activation up to cytotoxic concentration in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in either the initial plate incorporation assay or the independent repeat experiment using the pre-incubation method. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.