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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:
27 May - 25 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate
EC Number:
276-957-5
EC Name:
7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate
Cas Number:
72869-86-4
Molecular formula:
C23H38N2O8
IUPAC Name:
7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Method

Target gene:
his operon (for S. typhimurium strains)
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:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First experiment: 50, 150, 500, 1501 and 5004 µg/plate with and without metabolic activation
Second experiment: 312, 624, 1247, 2493 and 4986 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen because the test substance was completely soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants.
Controls
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine (4-NPD); 2-Amino-Anthracene (2-AA)
Remarks:
+S9: 2-AA (1 µg/plate, TA100, TA102, TA1535, TA97a); B[a]P (20 µg/plate, TA98); -S9: sodium azide (1 µg/plate, TA100 and TA1535); 4-NPD (20 µg/plate, TA102, TA97a, TA98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: quadruplicates each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test substance is considered to have a mutagenic potential if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
Mean values, standard deviations and the increase factor of revertant induction were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: The determined values for the spontaneous revertants of the negative controls and all positive control values were in the range of historical data.

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 4 plates ± standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA102

TA1535

TA97a

TA98

-

0 (H2O)

110 ± 12.0

264 ± 47.0

6 ± 1.9

105 ± 4.2

7 ± 1.4

-

0 (DMSO)

97 ± 6.8

296 ± 21.6

6 ± 2.1

96 ± 13.4

10 ± 1.0

-

50

97 ± 14

307 ± 7

6 ± 1

104 ± 21

4 ± 1

-

150

96 ± 17

310 ± 25

6 ± 1

105 ± 2

7 ± 3

-

500

102 ± 9

315 ± 14

7 ± 4

89 ± 18

7 ± 1

-

1501

108 ± 1

268 ± 36

8 ± 3

107 ± 5

7 ± 4

-

5004

99 ± 10

275 ± 49

6 ± 2

92 ± 6

7 ± 3

Positive controls,
-S9 mix

Name

NaN3

4-NPD

NaN3

4-NPD

4-NPD

Concentrations

[μg/plate]

1

20

1

20

20

Mean No. of colonies/plate

(average of 4 ± SD)

630 ± 44

1285 ± 185

374 ± 18

445 ± 35

278 ± 56

+

0 (H2O)

101 ± 15.8

345 ± 20.0

7 ± 1.5

97 ± 16.7

5 ± 2.8

+

0 (DMSO)

108 ± 0.8

305 ± 51.7

9 ± 4.2

90 ± 13.3

7 ± 3.1

+

50

107 ± 2

304 ± 21

6 ± 3

109 ± 3

6 ± 1

+

150

109 ± 3

276 ± 33

7 ± 3

109 ± 7

6 ± 1

+

500

100 ± 6

309 ± 17

8 ± 3

111 ± 10

8 ± 2

+

1501

94 ± 15

315 ± 59

4 ± 2

99 ± 15

6 ± 2

+

5004

118 ± 21

330 ± 40

13 ± 4

96 ± 15

7 ± 2

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

B[a]P

Concentrations

[μg/plate]

1

1

1

1

20

Mean No. of colonies/plate

(average of 4 ± SD)

524 ± 102

1093 ± 53

105 ± 12

319 ± 22

99 ± 9

NaN3: Sodium azide

4-NPD: 4-Nitro-1,2-phenylene diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene

 

Table 2. Test results of Experiment 2 (preincubation)

With or without S9 mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 4 plates ± standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA102

TA1535

TA97a

TA98

-

0 (H2O)

121 ± 10.0

299 ± 52.4

12 ± 3.2

103 ± 4.1

10 ± 0.0

-

0 (DMSO)

93 ± 7.1

274 ± 45.0

12 ± 1.9

110 ± 9.0

10 ± 3.7

-

312

106 ± 12

282 ± 22

8 ± 5

92 ± 14

11 ± 6

-

624

91 ± 9

232 ± 16

7 ± 1

110 ± 19

11 ± 2

-

1247

110 ± 30

255 ± 46

4 ± 4

109 ± 10

10 ± 3

-

2493

124 ± 12

192 ± 9

8 ± 3

114 ± 11

13 ± 4

-

4986

100 ± 7

188 ± 18

9 ± 6

98 ± 9

10 ± 2

Positive controls,
-S9 mix

Name

NaN3

4-NPD

NaN3

4-NPD

4-NPD

Concentrations

[μg/plate]

1

20

1

20

20

Mean No. of colonies/plate

(average of 4 ± SD)

386 ± 37

1127 ± 178

316 ± 17

903 ± 73

978 ± 72

+

0 (H2O)

97 ± 10.9

217 ± 12.9

10 ± 3.0

98 ± 7.4

14 ± 3.4

+

0 (DMSO)

109 ± 18.1

290 ± 30.2

10 ± 3.4

111 ± 2.1

11 ± 2.6

+

312

109 ± 8

279 ± 34

8 ± 2

94 ± 10

11 ± 2

+

624

106 ± 7

220 ± 15

5 ± 3

104 ± 7

11 ± 2

+

1247

101 ± 10

218 ± 21

12 ± 2

104 ± 9

13 ± 3

+

2493

95 ± 3

250 ± 51

10 ± 6

102 ± 3

11 ± 3

+

4986

92 ± 6

235 ± 29

8 ± 2

103 ± 4

7 ± 2

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

B[a]P

Concentrations

[μg/plate]

1

1

1

1

20

Mean No. of colonies/plate

(average of 4 ± SD)

499 ± 34

1345 ± 115

124 ± 12

826 ± 63

50 ± 5

NaN3: Sodium azide

4-NPD: 4-Nitro-1,2-phenylene diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene

Applicant's summary and conclusion