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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:
From 14 July 2004 to 19 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-nitrophenyl)amino]ethanol
EC Number:
225-555-8
EC Name:
2-[(2-nitrophenyl)amino]ethanol
Cas Number:
4926-55-0
Molecular formula:
C8H10N2O3
IUPAC Name:
2-[(2-nitrophenyl)amino]ethanol
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot Code - 17
- Expiration date of the lot/batch: 11 December 2005
- Purity test date: 11 December 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from exposure to light and moisture
- Stability under test conditions: the test article was stable in DMSO at ≤ -20°C for at least 12 and 14 days at 0.052 and 513 mg/mL, respectively
- Solubility and stability of the test substance in the solvent/vehicle:The test article was insoluble in water from 10 to 50 mg/mL, the concentrations tested. The test article formed a soluble and clear solution in dimethyl sulfoxide (DMSO) at approximately 500 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The vehicle used to deliver the test item to the test system was dimethyl sulfoxide (DMSO, CAS No.
67-68-5), obtained from Fisher Scientific. The 100 mg/mL concentration was prepared by diluting
the test article in dimethyl sulfoxide. Serial dilutions were prepared by diluting in dimethyl
sulfoxide. Test article dilutions were prepared immediately before use and delivered to the test
system at room temperature under yellow light. Dosing solutions were adjusted to compensate for
the purity (99.9%) of the test article.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: Wp2 uvrA (pKM101)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice
Test concentrations with justification for top dose:
In the initial mutagenicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 1800 or at 5000 μg per plate. Based on the findings of the initial mutagenicity assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article was insoluble in water from 10 to 50 mg/mL, the concentrations tested. The test article formed a soluble and clear solution in dimethyl sulfoxide (DMSO) at approximately 500 mg/mL, the maximum concentration tested. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): 10E9 per mL

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): After incubation at 2-8°C for 48 to 72 hours

NUMBER OF REPLICATIONS: duplicate per conditions in the pre screen test and triplicate per condition for the Main Test

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Counted either entirely by automated colony counter or entirely by hand unless the assay was
the preliminary toxicity assay or the plate exhibited toxicity.

NUMBER OF CELLS EVALUATED: not specified


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; Bacteria background lawn
- Any supplementary information relevant to cytotoxicity: using a dissecting microscope.Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the table 1 below.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA (pKM101) were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.
Statistics:
No statistical analysis was specified in the report

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the initial mutagenicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 1800 or at 5000 μg per plate. Based on the findings of the initial mutagenicity assay, the maximum dose plated in the confirmatory
mutagenicity assay was 5000 μg per plate.
A positive mutagenic response was observed with tester strain TA98 (8.0-fold, maximum increase) in the presence of S9 activation. No positive mutagenic response was observed with any of the remaining tester strains in the presence of S9 activation and with any of the tester strains in the absence of S9 activation.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See table 4 below

Any other information on results incl. tables

 

TABLE 2 : Summary of Results – Initial Toxicity-Mutation Assay Table

Test Article Id

: GTS03975

Study Number

: AA95CG.508.BTL

ExperimentNo : B1

AverageRevertantsPer Plate ± Standard Deviation LiverMicrosomes:

None

Dose (µg/plate)

TA98

 

TA100

 

TA1535

 

TA1537

 

WP2uvrA

 

 

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Vehicle

24

0

136

15

28

1

8

1

247

4

2.5

20

3

139

17

22

9

2

1

224

6

7.5

14

7

111

7

18

5

10

4

242

22

25

15

6

130

8

17

0

2

1

226

29

75

17

1

106

9

23

3

5

0

219

9

200

14

6

137

5

17

11

6

1

196

16

600

16

6

118

2

28

4

7

1

149

3

1800

20

1

88

8

23

3

7

1

89

4

5000

0

0

0

0

0

0

0

0

0

0

Positive

403

44

482

65

379

51

753

297

946

74

LiverMicrosomes:

RatliverS9

Dose (µg/plate)

TA98

 

TA100

 

TA1535

 

TA1537

 

WP2uvrA

 

 

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Vehicle

29

0

139

7

15

4

12

2

289

6

2.5

32

6

124

5

18

4

5

3

291

34

7.5

31

0

146

13

17

1

5

3

252

8

25

24

8

164

14

12

5

8

1

296

14

75

29

6

157

21

24

3

8

5

251

24

200

41

11

148

4

11

1

4

0

180

3

600

72

7

156

24

15

4

9

3

146

22

1800

233

11

154

21

14

2

17

3

87

8

5000

0

0

0

0

0

0

0

0

0

0

Positive

1249

96

891

73

97

4

122

16

1377

208

Vehicle = Vehicle Control

Positive = Positive Control (50 µL plating aliquot) Plating aliquot: 50 µL

 

 

 

TABLE 3 : Summary of Results – Confirmatory Mutagenicity Assay Table 22

Test Article Id

: GTS03975

Study Number

: AA95CG.508.BTL

ExperimentNo : B2

AverageRevertantsPer Plate ± StandardDeviationLiverMicrosomes:

None

Dose (µg/plate)

TA98

 

TA100

 

TA1535

 

TA1537

 

WP2uvrA

 

 

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Vehicle

27

1

152

7

24

8

4

1

278

11

25

16

4

135

18

16

6

6

3

241

28

75

17

2

119

7

21

5

5

2

232

23

200

14

5

124

22

20

8

5

3

224

9

600

22

2

114

12

19

5

5

3

194

19

1200

18

4

135

6

21

7

4

1

180

15

1800

17

5

125

11

22

6

4

1

163

17

5000

0

0

0

0

0

0

0

0

0

0

Positive

456

36

529

56

429

16

1199

39

943

4

LiverMicrosomes:

RatliverS9

Dose (µg/plate)

TA98

 

TA100

 

TA1535

 

TA1537

 

WP2uvrA

 

 

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Vehicle

39

8

150

19

21

2

7

3

268

11

25

35

3

154

32

16

3

11

5

284

30

75

38

6

122

19

20

3

8

2

295

13

200

37

4

171

9

17

1

9

3

268

6

600

57

9

135

10

17

1

9

3

255

13

1200

69

14

160

8

17

4

14

4

210

14

1800

169

82

152

13

16

6

10

2

183

9

5000

0

0

0

0

0

0

0

0

0

0

Positive

831

40

610

62

110

3

154

15

1420

70

Vehicle = Vehicle Control

Positive = Positive Control (50 µL plating aliquot) Plating aliquot: 50 µL

 

Table 4 :HistoricalData

Historical NegativeandPositive Control Values2001 – 2003

 

revertantsper plate

 

Strain

 

Control

Activation

None

Rat Liver

Mean

SD

Min

Max

Mean

SD

Min

Max

 

TA98

Neg

15

5

5

49

20

7

5

49

Pos

218

165

30

1981

695

385

40

2294

 

TA100

Neg

159

34

76

262

167

36

80

271

Pos

606

140

271

2373

956

438

262

2922

 

TA1535

Neg

15

6

3

46

13

5

2

50

Pos

344

140

16

1050

146

80

11

2246

 

TA1537

Neg

7

3

1

23

7

3

1

28

Pos

639

386

13

2351

131

135

12

2021

WP2uvrA(pKM101)

Neg

189

34

131

297

198

39

132

301

Pos

1169

395

470

2196

1186

326

380

1835

SD=standard deviation; Min=minimum value; Max=maximum value;Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone);Pos=positive control

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, test article GTS0375 was concluded to be positive in Salmonella typhimurium tester strain TA98 in the presence of Aroclor-induced rat liver S9.
Executive summary:

In this GLP-compliant study, The test item HC Yellow No. 2 was investigated for the induction of gene mutations in Salmonella typhimurium and Escherichia coli (Ames test) according to OECD 471 method.

The concentrations used in this study were :

initial experiment: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg/plate without and with S9-mix

confirmatory experiment: 25, 75, 200, 600, 1200, 1800 and 5000 μg/plate without and with S9-mix

Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. The initial mutagenicity assay both without and with S9-mix was used to establish the dose range for the confirmatory assay and to provide a preliminary mutagenicity evaluation. The condition of the bacterial background lawn was evaluated for evidence of toxicity of the test item. Both experiments were performed without and with S9-mix using the preincubation method. Negative and positive controls were in accordance with the OECD guideline.

Results

In both the initial and the confirmatory experiment without and with S9-mix no precipitation occurred whereas toxicity was seen at the highest dose tested, 5000 μg/plate. A biologically relevant and more or less dose dependent increase in the number of revertant colonies was observed in strain TA98 after exposure to the test item in the presence of S9-mix. No mutagenic response was observed in the other tester strains in the presence of S9-

mix and with any of the tester strains in the absence of S9-mix.

Conclusion

Under the experimental conditions used the test item was mutagenic in this gene mutation tests in bacteria.