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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:
24 Aug - 24 Sep 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ß-naphthoflavone (80/100 mg/kg bw/d, oral)
Test concentrations with justification for top dose:
Preliminary toxicity study:
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for TA100

First experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA98
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA100, TA1535, TA102 and TA1537
5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA1535
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for TA100, TA102, TA98 and TA1537

Second experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for all strains
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for all strains
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene (2AA); 1,8- dihydroxyanthraquinon (DANTHRON)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in experiment 1 and 2

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results
Species / strain:
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:
starting at 500 µg/plate with and without S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The test material was toxic at 5000 µg/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies in all of the tester strains both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 500 µg/plate. Generally, the test material induced a greater toxic response after the addition of S9-mix. The sensitivity of the bacterial strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit.

Any other information on results incl. tables

Table 1. Test results of experiment 1

With or without S9-mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

128

20

247

24

7

0 (DMSO)

114 ± 13.7

30 ± 3.5

317 ± 36.4

28 ± 3.5

13 ± 2.3

15

NT

NT

NT

25 ± 4.0

NT

50

109 ± 4.4

19 ± 5.5

278 ± 3.6

22 ± 3.2

11 ± 2.9

150

112 ± 12.0

20 ± 2.6

281 ± 31.8

31 ± 6.5

12 ± 4.5

500

105 ± 2.1

15 ± 5.5

279 ± 22.2

28 ± 2.0

11 ± 1.0

1500

67 ± 6.1

5 ± 1.7

107 ± 12.1

10 ± 2.9

5 ± 4.7

5000

0

0

0T

0

0

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

(μg/plate)

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

539 ± 64.2

222 ± 39.2

751 ± 184.5

171 ± 19.0

784 ± 250.9

+

0

143

22

310

-

10

+

0 (DMSO)

145 ± 3.0

12 ± 3.5

310 ± 28.7

39 ± 1.7

16 ± 2.6

+

5

119 ± 9.8

12 ± 0.6

284 ± 63.4

40 ± 3.1

17 ± 7.8

+

15

118 ± 6.4

17 ± 4.5

318 ± 51.8

27 ± 8.1

15 ± 6.7

+

50

120 ± 14.7

10 ± 1.7

333 ± 21.7

29 ± 2.5

9 ± 2.1

+

150

98 ± 4.7

13 ± 2.6

296 ± 5.8

31 ± 4.0

10 ± 2.0

+

500

85 ± 6.1

6 ± 2.6

0V

12 ± 3.8

0V

+

1500

0V

0V

0V

0V

0T

+

5000

NT

0T

NT

NT

NT

Positive controls, +S9

Name

2-AA

2-AA

DAN

B[a]P

2-AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1770 ± 182.4

192 ± 16.2

707 ± 65.6

255 ± 6.7

685 ± 40.8

DMSO: Dimethylsulphoxide

NT: Not tested at this dose level

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

MMC: Mitomycin C

B[a]P: Benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

T: Toxic

V: Very weak lawn

Table 2. Test results of experiment 2.

With or without S9-mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

145

20

306

32

12

0 (DMSO)

147 ± 5.7

20 ± 1.5

339 ± 3.2

33 ± 4.0

12 ± 5.9

15

127 ± 17.7

18 ± 0.6

295 ± 23.0

38 ± 6.5

12 ± 4.9

50

146 ± 7.6

18 ± 2.6

300 ± 16.1

28 ± 5.3

5 ± 1.7

150

124 ± 4.5

15 ± 0.6

297 ± 29.4

22 ± 5.0

9 ± 4.6

500

110 ± 26.5

10 ± 0.6

310 ± 14.9

22 ± 5.5

6 ± 3.1

1500

63 ± 5.6

4 ± 2.1

158 ± 18.7

9 ± 3.5

6 ± 3.6

5000

0

2 ± 1.5

0

1 ± 1.2

4 ± 3.2S

Positive controls, –S9

Name

ENNG

ENNG

MMC

4-NQO

9-AA

Concentrations

(μg/plate)

3

5

0.5

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

471 ± 47.3

144 ± 20.9

1054 ± 39.6

139 ± 52.3

1032 ± 104.8

+

0

90

-

-

-

-

+

0 (DMSO)

107 ± 9.5

17 ± 5.0

302 ± 17.1

32 ± 3.5

10 ± 2.3

+

5

103 ± 3.8

15 ± 0.6

302 ± 34.8

28 ± 2.6

10 ± 4.4

+

15

100 ± 4.6

11 ± 2.9

310 ± 9.2

28 ± 4.6

8 ± 0.6

+

50

88 ± 20.3

16 ± 5.7

289 ± 12.9

38 ± 7.1

8 ± 0.6

+

150

85 ± 7.6

15 ± 4.2

265 ± 11.1

29 ± 1.5

3 ± 1.0

+

500

59 ± 2.5

11 ± 0.6

0S

10 ± 7.4S

0V

+

1500

21 ± 4.0V

2 ± 2.1S

0V

9 ± 3.1S

0T

Positive controls, +S9

Name

2-AA

2-AA

DAN

B[a]P

2-AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

1277 ± 77.7

124 ± 7.4

727 ± 165.4

188 ± 13.2

169 ± 12.0

DMSO: Dimethylsulphoxide

NT: Not tested at this dose level

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

MMC: Mitomycin C

B[a]P: Benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

S: Sparse lawn

T: Toxic

V: Very weak lawn

Table 3. Historical control data

positive control values up to and including 1997
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 117 120 28 33 24 18 21 25 10 11 20 21 264 283
SD 25 22 7 7 5 4 7 6 2 2 5 s 30 33
Min 61 63 13 14 10 11 6 11 4 5 12 11 216 205
Max 196 177 56 52 39 39 41 50 17. 20 .39 40 339 343
Values 1603 863 1047 837 948 718 309 190 938 703 634 436 197 100
vehicle control values up to and including 1997
Strain  TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 613 949 203 420 466 291 484 454 812 266 834 734 785 705
SD 176 190 57 113 208 64 146 131 201 90 197 211 172 127
Min 277 412 127 198 163 139 190 212 161 123 342 225 540 511
M ax 1126 1315 698 757 1005 516 799 915 1149 718 1209 1089 1188 1090
Values 195 195 194 195 180 181 93 92 182 183 162 157 76 72
positive control values up to and including 1998
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 124 125 31 36 26 18 26 28 11 14 23 25 262 285
SD 16 16 6 6 5 4 6 5 3 3 4 4 31 32
Min 73 81 15 22 12 10 15 18 5 6 13 13 191 231
Max 173 170 45 54 38 37 36 36 20 23 34 41 304 363
Values 342 339 337 340 341 331 16 16 323 325 287 285 20 22
vehicle control values up to and including 1998
Strain TA100 TA98 TA1535 TA1538 TA1537 WP2uvrA TA102
S9-Mix +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9
Mean 556 1209 143 472 375 260 403 556 1052 298 851 837 863 747
SD 114 318 24 119 173 64 93 197 259 106 202 223 127 116
Min 349 681 101 198 112 105 305 344 173 123 415 260 646 591
Max 1147 2740 225 733 1143 601 604 1073 1912 652 1486 1380 1096 1096
Values 141 141 140 143 142 141 12 12 142 141 138 138 24 25

SD = standard deviation

Min = minimal value

Max = maximal value

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and TA102) with and without metabolic activation tested.