Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 - 22 Dec 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
June 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Niedersächsisches Umweltministerium, Hannover, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with phenobarbital (i.p.) and β-naphthoflavone (oral)
Test concentrations with justification for top dose:
Experiment 1:
16, 50, 160, 500 and 1600 µg/plate with and without metabolic activation

Experiment 2:
16, 50, 160, 500 and 1600 µg/plate with and without metabolic activation in TA 97a
50, 160, 500, 1600 and 5000 µg/plate with and without metabolic activation in TA 98 and TA 102
16, 50, 160, 500 and 1600 µg/plate without metabolic activation in TA 100 and TA 1535
50, 160, 500, 1600 and 5000 µg/plate with metabolic activation in TA 100 and TA 1535
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: ICR 191 acridine mutagen dihydrochloride (ICR 191), 2-aminoanthracene (2-AA), 4-nitro-1,2-phenylenediamine (4-NOPD), nitrofurantoine (NF), cumene hydroperoxide (CUHP), dantron
Remarks:
+S9: 2-AA (2.0 µg/plate, TA97a, TA98, TA100 and TA1535); dantron (30 µg/plate, TA102); -S9: ICR 191 (0.5 µg/plate, TA97a); NaN3 (0.25 µg/plate, TA1535); 4-NOPD (0.5 µg/plate, TA98); NF (0.2 µL/plate, TA100); CUHP (100 µg/plate, TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduced bacterial background lawn
Evaluation criteria:
Since a reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background Iawn were not included into evaluation procedures.
The test substance is considered as mutagenic if there is a concentration effect relationship and the induction rate is ≥ 2.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: -S9: at 1600 µg/plate; Exp. 2: -S9: at 1600 µg/plate, +S9: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 2: -S9 and +S9: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: -S9: at 1600 µg/plate; Exp. 2: -S9: at 1600 µg/plate, +S9: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
Exp. 2: -S9 and +S9: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1 and 2: -S9 and +S9: at 1600 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA1535

TA100

TA102

TA97a

TA98

-

0 (DMSO)

24 ± 6.1

151 ± 42.2

427 ± 73.1

391 ± 9.9

34 ± 0.6

-

16

17 ± 4.4

143 ± 6.1

465 ± 65.9

441 ± 72.6

26 ± 7.8

-

50

19 ± 7.5

141 ± 5.9

400 ± 24.0

422 ± 7.1

28 ± 4.0

-

160

8 ± 3.0

145 ± 12.9

435 ± 54.0

386 ± 11.7

28 ± 1.7

-

500

14 ± 2.0

119 ± 20.2

388 ± 14.5

354 ± 15.6

27 ± 3.2

-

1600

t

t

177 ± 18.7

t

18 ± 8.7

Positive controls, –S9

Name

NaN3

NF

CUHP

ICR 191

4-NOPD

Concentration

[μg/plate]

0.25

0.2

100

0.5

0.5

Induction rate

9.0

2.4

> 2.8

> 3.1

3.6

+

0 (DMSO)

14 ± 2.1

130 ± 2.1

538 ± 57.5

315 ± 66.2

37 ± 4.5

+

16

8 ± 4.0

136 ± 2.0

454 ± 49.2

371 ± 23.4

34 ± 2.6

+

50

12 ± 4.0

137 ± 11.0

406 ± 38.2

347 ± 27.2

35 ± 2.1

+

160

10 ± 5.5

140 ± 15.5

443 ± 46.3

358 ± 62.4

35 ± 7.6

+

500

11 ± 2.5

127 ± 8.4

439 ± 41.6

262 ± 86.7

39 ± 1.2

+

1600

10 ± 5.0

75 ± 9.1

337 ± 23.8

t

51 ± 14.6

Positive controls, +S9

Name

2-AA

2-AA

Dantron

2-AA

2-AA

Concentration

[μg/plate]

2.0

2.0

30

2.0

2.0

Induction rate

9.8

> 9.3

> 2.2

> 3.8

> 32.1

NaN3: sodium azide

4-NOPD: 4-nitro-1,2-phenylene-diamine

2-AA: 2-aminoanthracene

ICR 191: ICR 191 acridine mutagen dihydrochloride

NF: nitrofurantoine

CUHP: cumene hydroperoxide

t: toxic (reduced background lawn; not counted)

Table 2. Test results of Experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA1535

TA100

TA102

TA97a

TA98

-

0 (DMSO)

15 ± 4.7

118 ± 8.3

354 ± 34.8

368 ± 72.3

18 ± 3.0

-

16

11 ± 1.5

103 ± 14.2

-

467 ± 47.3

-

-

50

8 ± 4.0

122 ± 25.3

338 ± 33.2

419 ± 7.1

30 ± 3.6

-

160

8 ± 2.0

110 ± 4.6

315 ± 18.3

396 ± 17.1

28 ± 2.6

-

500

15 ± 6.6

101 ± 15.0

312 ± 24.2

392 ± 100.1

28 ± 5.7

-

1600

t

t

151 ± 22.6

t

36 ± 3.6

-

5000

-

-

t

-

t

Positive controls, –S9

Name

NaN3

NF

CUHP

ICR 191

4-NOPD

Concentration

[μg/plate]

0.25

0.2

100

0.5

0.5

Induction rate

9.9

3.2

> 3.4

> 3.3

5.3

+

0 (DMSO)

13 ± 3.5

134 ± 4.9

488 ± 107.0

338 ± 38.8

34 ± 6.1

+

16

-

-

-

368 ± 31.4

-

+

50

10 ± 3.2

107 ± 22.5

279 ± 26.0

354 ± 11.1

32 ± 4.0

+

160

11 ± 3.5

97 ± 11.1

314 ± 16.4

311 ± 21.2

35 ± 0.6

+

500

9 ± 2.1

105 ± 22.3

294 ± 8.3

329 ± 12.0

34 ± 6.1

+

1600

8 ± 2.0

75 ± 4.2

192 ± 27.2

t

50 ± 7.0

+

5000

t

t

t

-

t

Positive controls, +S9

Name

2-AA

2-AA

Dantron

2-AA

2-AA

Concentration

[μg/plate]

2.0

2.0

30

2.0

2.0

Induction rate

6.3

> 8.9

> 2.5

> 3.6

> 35.3

NaN3: sodium azide

4-NOPD: 4-nitro-1,2-phenylene-diamine

2-AA: 2-aminoanthracene

ICR 191: ICR 191 acridine mutagen dihydrochloride

NF: nitrofurantoine

CUHP: cumene hydroperoxide

t: toxic (reduced background lawn; not counted)

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 97a, TA 98, TA 100, TA 102 and TA 1535) tested with and without metabolic activation up to cytotoxic concentrations or 5000 µg/plate.
Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2001). In this study the substance was not mutagenic in any of the five strains (TA 97a, TA 98, TA100, TA 102 and TA 1535) tested with and without metabolic activation up to cytotoxic concentrations or 5000 µg/plate.