Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation assay has been performed on test item tetramethrin according to OECD guideline no. 471. The test item induces increases in the number of revertant colonies in TA100 tester strain, in the presence of S9 metabolism. However, these increases were two-fold greater than the control value only in the first experiment and the dose response was not reproduced. The results obtained preclude making a definite judgement about the activity of the test item.

An increase in mutant frequency above the Global Evaluation Factor was observed after continuous 24-h treatment with tetramethrin in the absence of S9 metabolism in an in vitro mammalian cell gene mutation test (mouse lymphoma assay). However, this increase occurred in the presence of significant cytotoxicity (25% RS). On the basis of this results, the criteria for a clear mutagenic effect were not met and the outcome was regarded as equivocal.

An in vitro mammalian chromosome aberration test has been performed on test item tetramethrin according to OECD guideline no. 473. Tetramethrin does not induce chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment in the presence or absence of metabolic activation, under the reported experimental conditions.

A DNA damage and repair study with E.coli W3110/polA+ and p3478/polA- was performed with tetramethrin but did not show any DNA damaging potential.

Based on an overall weight of evidence by four in vitro and one in vivo mutagenicity studies, it is concluded that tetramethrin does not require classification for mutagenicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: uvrB,rfa,pKM101,pAQ1 as genetic markers beside histidine deficiency
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue homogenate
Test concentrations with justification for top dose:
The test item TETRAMETHRIN was found to be soluble in DMSO at a concentration of 300 mg/ml. Since 100 µl of the test item solution are used in the preparation of each plate, this permitted a maximum concentration of 5000 µl/plate to be used in the toxicity test.
No signs of toxicity were observed at any dose-level tested, in any tester strain, in the absence or presence of S9 metabolic activation. On the basis of these results a maximum concentration of 5000 µg/plate was selected for the Main Assay I with all tester strains. Precipitation of the test item was observed at 5000 µg/plate.
Two main experiments were performed.
In Main Assay I, using the plate incorporation method, the test item was assayed at a maximum dose-level of 5000 µg/plate and at four lower dose-levels, separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate.
in Main Assay II the same dose-levels used in Main Assay I were employed. A pre-incubation step was included in all treatments with the exception of testing TA100 in the presence of S9 metabolism. With this tester strain the test item was assayed (in the presence of S9 metabolism) under the same experimental conditions employed in Main Assay I.
Vehicle / solvent:
Dimethylsulphoxide (DMSO): (Fluka AG, batch 404161/1 13400).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cumene hydroperoxide
other: 2-Aminoanthracene, sterile distilled water
Details on test system and experimental conditions:
Preliminary toxicity test
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose-levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included.
Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Main experiments
Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix, and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate, and allowed to solidify prior to incubation.

The overlay mixture was composed as follows:
(i) Overlay agar (held at 45 °C) 2 ml
(ii) Test or control item solution 0.1 ml
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml
(iv) Bacterial suspension 0.1 ml

The second experiment was performed using a pre-incubation method with all tester strains with the exception of TA100 in the presence of S9 metabolism: this treatment was, instead, carried out using the plate incorporation method.
In the pre-incubation method the components were added in turn to an empty test-tube:
(i) Bacterial suspension 0.1 ml
(ii) Test item and control solution 0.05 ml
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml

The incubate was vortexed and placed at 37 °C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Incubation and scoring
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, the scoring was effected by counting the number of revertant colonies on each plate.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In order to confirm the results obtained with TA100, in Main Assay II the test item was assayed, with this tester strain in the presence of metabolic activation, under the same experimental conditions used in Main Assay I. The other treatments of Main Assay II included a pre-incubation step and used the same dose-levels as Main Assay I. Thinning of the background lawn was observed at the highest dose-level in all tester strains in the absence of S9 metabolism.
Increases in revertant colonies were again observed in TA100 in the presence of S9. However, these increases did not reach twice the control value at any dose-level assayed and were not concentration-related over the range tested.
Precipitation of the test item was observed at 5000 and 2500 µg/plate in both experiments.
The sterility of the S9 mix and the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Remarks on result:
other: without pre-incubation

TABLE 1 - WITHOUT METABOLIC ACTIVATION

STUDY NO.: 9324

SOLVENT: DMSO

EXPERIMENT: Toxicity test

Dose-level (µg/plate}

TA-1535
Rev/pl.

TA-1537
Rev/pl.

TA-98 Rev/pl.

TA-100
Rev/pl.

TA-102
Rev/pl.

Untreated

18

19

32

131

384

0.00

15

20

25

119

376

50.0

24

22

29

149

399

158

16

19

29

130

406

500

18

14

32

155

397

1580

13

16

29

145

390

5000

18

16

36

177

358

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 2 - WITH METABOLIC ACTIVATION

STUDY NO.: 9324

SOLVENT: DMSO

EXPERIMENT: Toxicity test

Dose-level (µg/plate)

TA-1535
Rev/pl.

TA-1537
Rev/pl.

TA-98 Rev/pl.

TA-100
Rev/pl.

TA-102
Rev/pl.

Untreated

12

23

38

159

409

0.00

15

24

37

134

417

50.0

12

21

40

138

407

158

13

22

39

152

447

500

16

17

40

221

470

1580

14

18

42

214

451

5000

12

20

41

229

487

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 3 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA1535                               Titre: 232

Dose-level    Without metabolic activ.   With metabolic activation

[pg/pl]       Plate counts   Mean S. E.       Plate counts      Mean S. E.

Untreated

24

16

19

20

2.3

19

14

16

16

1.5

0.00

20

19

19

19

0.3

20

17

19

19

0.9

313

18

18

22

19

1.3

17

16

19

17

0.9

625

21

16

21

19

1.7

15

13

16

15

0.9

1250

18

25

21

21

2.0

13

13

19

15

2.0

2500

20

24

19

21

1.5

19

15

16

17

1.2

5000

24

27

21

24

1.7

17

13

10

13

2.0

Regression analysis

Points

S9

Intercept

Slope

Corr.coeff.

t

P-value

1  3

-

4.396

0.0000

-0.01667

0.0441

0.96605

1- 4

 

4.351

0.0002

0.32072

1.0708

0.30944

1-5

-

4.388

0.0001

0.31501

1.1967

0.25279

1-6

 

4.380

0.0001

0.57959

2.8449

0.01170

1-3

+

4.348

-0.0008

-0.78438

3.3457

0.01232

1- 4

+

4.249

-0.0004

-0.58151

2.2603

0.04734

1-5

+

4.112

-0.0001

-0.21169

0.7809

0.44882

1-6

+

4.132

-0.0001

-0.47471

2.1575

0.04652

Positive and negative controls Treatment

S9         Plate counts             Mean     S. E.

Untreated                                 -            24 16 19                   20         2.3

Sodium Azide 1 pg/pl              -            647 667 626             647       11.8

DMSO           100 pl/pl             +            20 17 19                    19         0.9

2-Aminoanthracene 1 pg/pl    +            126 131 114             124        5.0

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 4 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324 SOLVENT: DMSO

Strain: TA1537                               Titre: 221

Dose-level  Without metabolic activ.    With metabolic activation

[µg/pl]       Plate counts   Mean S. E.       Plate counts  Mean S. E.

Untreated

18

17

20

18

0.9

25

18

25

23

2.3

0.00

18

17

18

18

0.3

19

25

20

21

1.9

313

16

13

15

15

0.9

20

18

20

19

0.7

625

18

21

17

19

1.2

24

18

20

21

1.8

1250

15

14

15

15

0.3

25

19

18

21

2.2

2500

17

15

14

15

0.9

24

24

23

24

0.3

5000

19

15

17

17

1.2

20

21

19

20

0.6

Regression analysis

Points

S9

Intercept

Slope

Corr.        coeff.

t

P-value

1-3

 

4.059

0.0002

0.17859

0.4802

0.64572

1- 4

 

4.150

-0.0002

-0.34834

1.1751

0.26716

1-5

 

4.106

-0.0001

-0.34012

1.3041

0.21483

1-6

 

4.037

0.0000

-0.01146

0.0458

0.96400

1-3

+

4.551

-0.0001

-0.11856

0.3159

0.76128

1-4

+

4.525

0.0000

-0.01818

0.0575

0.95527

1-5

+

4.461

0.0001

0.42601

1.6978

0.11335

1-6

+

4.557

0.0000

0.04910

0.1966

0.84659

Positive and negative controls

Treatment                                 S9 Plate counts    Mean S.E.

DMSO

100

µl/p1

-

18

17

18

18

0.3

9-Aminoacridine

50

µg/p1

-

167

143

186

165

12.4

DMSO

100

Al/p1

+

19

25

20

21

1.9

2-Aminoanthracene

1

µg/p1

+

96

105

101

101

2.6

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 5 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324 SOLVENT: DMSO

Strain: TA102                                Titre: 269

Dose-level    Without metabolic activ.    With metabolic activation

[µg/pl]       Plate counts   Mean S. E.      late counts   Mean S. E.

Untreated

359

374

386

373

7.8

414

407

395

405

5.5

0.00

398

364

381

381

9.8

387

421

400

403

9.9

313

370

393

362

375

9.3

446

431

429

435

5.4

625

365

376

390

377

7.2

454

456

447

452

2.7

1250

384

392

389

388

2.3

483

472

475

477

3.3

2500

380

396

371

382

7.3

457

421

432

437

10.7

5000

396

366

360

374

11.1

484

479

457

473

8.3

Regression analysis

Points

S9

Intercept

Slope

Corr.        coeff.

t

P-value

1-3

-

19.482

-0.0002

-0.12630

0.3369

0.74609

1- 4

-

19.396

0.0002

0.28166

0.9283

0.37512

1-5

-

19.452

0.0001

0.18155

0.6657

0.51726

1-6

 

19.513

0.0000

-0.10362

0.4167

0.68241

1-3

+

20.129

0.0019

0.89109

5.1949

0.00126

1  4

+

20.270

0.0013

0.91902

7.3719

0.00002

1- 5

+

20.751

0.0002

0.35048

1.3493

0.20028

1  6

+

20.785

0.0002

0.53459

2.5303

0.02227

 

 

 

Positive and negative controls

treatment

S9

Plate counts

Mean

S. E.

DMSO

100 µl/p1

-

398  364   381

381

9.8

Cumene hydroperoxide

100 µg/p1

-

1254 1219 1181

1218

21.1

DMSO

100 µl/p1

+

387 421  400

403

9.9

2-Aminoanthracene

10 µg/pl

+

1528  1496 1581

1535

24.8

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 6 - Experiment I       - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA98                                    Titre: 242

Dose-level    Without metabolic activ.    With metabolic activation

[µg/pL]       Plate counts   Mean S.E.      Plate counts   Mean S.E.

Untreated

32

31

29

31

0.9

40

37

36

38

1.2

0.00

29

31

27

29

1.2

40

37

38

38

0.9

313

28

30

31

30

0.9

36

41

39

39

1.5

625

31

31

28

30

1.0

47

38

38

41

3.0

1250

29

27

28

28

0.6

40

39

38

39

0.6

2500

30

31

27

29

1.2

36

37

37

37

0.3

5000

30

29

36

32

2.2

40

37

39

39

0.9

Regression analysis

Points

S9

Intercept

Slope

Corr.   coeff.

t

P-value

1-3

 -

5.388

0.0001

0.27288

0.7505

0.47744

1-4

 -

5.445

-0.0001

-0.27746

0.9132

0.38260

1 -5

 -

5.413

0.0000

-0.07646

0.2765

0.78652

1-6

 -

5.375

0.0000

0.35336

1.5109

0.15031

1-3

+

6.165

0.0003

0.35163

0.9938

0.35343

1-4

+

6.231

0.0001

0.12475

0.3976

0.69927

1  5

+

6.286

-0.0001

-0.30385

1.1499

0.27089

1-6

+

6.246

0.0000

-0.14440

0.5837

0.56755

Positive and negative

controls Treatment           S9   Plate counts  Mean S. E.


DMSO

100

µl/p1

 

-

29

31

27

29

1.2

2-Nitrofluorene

2µl/p1

 

-

197

198

184

193

4.5

DMSO

100  

µl/p1

 

+

40

37

38

38

0.9

2-Aminoanthracene

1 

µl/pl

 

+

574

621

619

605

15.3

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 7 -Experiment I      - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA100                               Titre: 226

[µg/pl]

Plate counts

Mean

S.   E.

Plate counts

Mean

S.E.

Untreated

135

139

127

134

3.5

143

134

139

139

2.6

0.00

123

133

136

131

3.9

135

140

134

136

1.9

313

149

156

143

149

3.8

198

203

213

205

4.4

625

171

176

174

174

1.5

254

272

270

265

5.7

1250

177

189

195

187

5.3

308

294

280

294

8.1

2500

143

154

141

146

4.0

286

268

261

272

7.4

5000

188

187

158

178

9.8

282

300

302

295

6.4

Regression analysis

Points

S9

Intercept

Slope

Corr.coeff.

t

P-value

1-3

-

11.400

0.0028

0.96353

9.5269

0.00003

1-4

-

11.645

0.0018

0.92647

7.7842

0.00001

1-5

-

12.352

0.0002

0.18444

0.6766

0.51051

1-6

-

12.339

0.0002

0.39015

1.6949

0.10946

1-3

+

11.782

0.0074

0.99120

19.8166

0.00000

1-4

+

12.555

0.0042

0.91682

7.2607

0.00003

1-5

+

13.706

0.0016

0.69098

3.4465

0.00434

1-6

+

14.334

0.0007

0.63087

3.2524

0.00500

Treatment

S9

Plate counts

Mean

S.   E.

Untreated

-

135   139  127

134

3.5

Sodium Azide 1 µg/pl

-

954   977  926

952

14.7

DMSO 100 µl/pl

+

135   140  134

136

1.9

2-Aminoanthracene 1 µg/pl

+

1194 1159 1165

1173

10.8

 

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 8 - Experiment II       - Preincubation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA1535, Titre: 231

Dose-level[µg/pl]  Without metabolic activation With metabolic activation
Plate counts Mean s.e. Plate counts Mean s.e.
Untreated  15 18 20 18 1,5 14 16 12 14 1,2
0.00  19 18 12 16 2,2 17 15 11 14 1,8
313 17 13 12 14 1,5 10 18 19 16 2,8
625 19 17 18 18 0,6 13 19 15 16 1,8
1250 20 19 16 18 1,2 18 11 12 14 2,2
2500 18 15 14 16 1,2 19 11 10 13 2,8
5000 14 15 19 * 16 1,5 12 10 11 11 0,6

Regression analysis:

Points S9  Intercept Slope Corr. Coeff t P-value
1 - 3 - 3,888 0,0004 0,25197 0,6889 0,51307
1 - 4 - 3,898 0,0003 0,41431 1,4395 0,18056
1 - 5 - 4,025 0,0000 0,05840 0,2109 0,83623
1 - 6 - 4,045 0,0000 -0,02969 0,1188 0,90689
1 - 3 + 3,792 0,0003 0,17173 0,4612 0,65864
1 - 4 + 3,885 -0,0001 -0,11427 0,3637 0,72362
1 - 5 + 3,887 -0,0001 -0,21189 0,7817 0,44838
1 - 6 + 3,891 -0,0001 -0,43244 1,9184 0,07308

* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

 TABLE 9 - Experiment II  - Preincubation method -

STUDY NO.: 9324

SOLVENT: DMSO

Dose-level [µg/pl] Without metabolic activation
 
With metabolic activation
 
Plate counts Mean s.e. Plate counts Mean s.e.
Untreated 20 16 14 17 1,8 22 16 17 18 1,9
0,00 17 18 17 17 0,3 21 19 18 19 0,9
313 11 18 17 15 2,2 18 20 22 20 1,2
625 18 15 18 17 1,0 23 20 21 21 0,9
1250 17 11 15 14 1,8 17 19 22 19 1,5
2500 19 11 13 14 2,4 20 15 17 17 1,5
5000 11 7 12 10 1,5 16 12 19 16 2,0

Regression analysis:
Points S9 Intercept Slope Corr. coeff. t P-value
1 - 3 - 4,081 -0,0001 -0,06265 0,1661 0,87279
1 - 4 - 4,125 -0,0003 -0,36073 1,2231 0,24935
1 - 5 - 4,078 -0,0001 -0,35658 1,3761 0,19203
1 - 6 - 4,108 -0,0002 -0,68025 3,7123 0,00189
1 - 3 + 4,382 0,0004 0,50432 1,5452 0,16621
1- 4 + 4,469 0,0000 -0,00721 0,0228 0,98227
1 - 5 + 4,523 -0,0001 -0,45447 1,8396 0,08877
1- 6 + 4,516 -0,0001 -0,62715 3,2207 0,00534

* Thinning of the background lawn

Positive and negative controls Treatment   S9 Plate counts Mean s.e.
DMSO 50 µl/pl - 17 18 17 17 0,3
9-Aminoacridine 50 µg/pl - 125 146 186 152 17,9
DMSO 50 µl/pl + 21 19 18 19 0,9
2-Aminoanthracene 1 µg/pl + 110 112 127 116 5,4

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium
TABLE 10 - Experiment II - Preincubation method -
STUDY NO.: 9324
SOLVENT: DMSO
Strain: TA102 Titre: 255
Dose-level [µg/pl] Without metabolic activation
  
With metabolic activation
  
Plate counts Mean s.e. Plate counts Mean s.e.
Untreated 359 346 385   363 11,5 392 416 420 409 8,7
0,00 369 376 382 376 3,8 427 399 407 411 8,3
313 379 388 394 387 4,4 404 424 418 415 5,9
625 373 389 376 379 4,9 415 422 397 411 7,4
1250 370 381 368 373 4,0 417 411 420 416 2,6
2500 401 388 392 394 3,8 435 427 410 424 7,4
5000 370 393 384 * 382 6,7 427 410 431 423 6,4
Regression analysis:
Points S9 Intercept Slope Corr. coeff. t P-value
1 - 3 - 19,462 0,0002 0,19337 0,5215 0,61814
1 - 4 - 19,530 -0,0001 -0,28647 0,9455 0,36668
1 - 5 - 19,420 0,0001 0,44488 1,7910 0,09659
1 - 6 - 19,490 0,0000 0,21205 0,8680 0,39825
1 - 3 + 20,305 0,0000 0,01375 0,0364 0,97200
1 - 4 + 20,291 0,0001 0,14742 0,4713 0,64751
1 - 5 + 20,271 0,0001 0,40922 1,6171 0,12986
1 - 6 + 20,315 0,0001 0,39399 1,7147 0,10571
Positive and negative treatment controls
S9 Plate counts Mean s.e.
DMSO 50 µl/pl - 369 376 382 376 3,8
Cumene hydroperoxide 100 µg/pl - 1066 1192 1064 1107 42,3
DMSO 50 µl/pl + 427 399 407 411 8,3
2-Aminoanthracene 20 µg/pl + 1615 1596 1600 1604 5,8
* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 11 - Experiment II

STUDY NO.: 9324

SOLVENT: DMSO

 - Preincubation method -

Strain: TA98 Titre: 244
  
    
Dose-level [µg/pl] Without metabolic activation With metabolic activation
Plate counts Mean s.e. Plate counts mean s.e.
Untreated   37 30 34 34 2,0 39 44 40 41 1,5
0.00   31 31 32 31 0,3 38 36 43 39 2,1
313 31 32 35 33 1,2 38 35 41 38 1,7
625 32 34 34 33 0,7 36 38 35 36 0,9
1250 31 29 34 31 1,5 38 42 36 39 1,8
2500 29 27 33 30 1,8 38 38 41 39 1,0
5000 26 25 29 * 27 1,2 43 38 37 39 1,9
Regression analysis:                
Points  S9 Intercept Slope Corr. coeff. t P-value
1 - 3 - 5,607 0,0003 0,57691 1,8687 0,10388
1 - 4 - 5,680 0,0000 -0,05669 0,1796 0,86109
1 - 5 - 5,713 -0,0001 -0,44214 1,7773 0,09890
1 - 6 - 5,724 -0,0001 -0,74260 4,4351 0,00042
1 - 3 + 6,250 -0,0003 -0,42210 1,2319 0,25776
1- 4 + 6,171 0,0000 -0,04192 0,1327 0,89707
1- 5 + 6,150 0,0000 0,13195 0,4800 0,63923
1- 6 + 6,154 0,0000 0,20438 0,8351 0,41594
Positive and negative control - TREATMENTS S9 Plate counts Mean s.e.
DMSO 50µl/pl - 31 31 32 31 0,3
2-Nitrofluorene 2 µg/pl - 191 194 154 180 12,9
DMSO 50 µl/pl + 38 36 43 39 2,1
2-Aminoanthracene 2 µg/pl + 698 666 649 671 14,4
* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 12 - Experiment II      - Preincubation method -

STUDY NO.: 9324

SOLVENT DMSO

Titre: 224
Strain: TA100
Dose-level [µg/pl] Without metabolic activation
Plate counts Mean S.e.
Untreated 133 140 137   137 2,0
0,00 130 126 133 130 2,0
313 134 152 153 146 6,2
625 140 161 166 156 8,0
1250 147 150 155 151 2,3
2500 145 138 150 144 3,5
5000 152 157 164 * 158 3,5
Regression analysis:
Points  S9 Intercept Slope Corr. coeff. t P-value
1 - 3 - 11,441 0,0017 0,78057 3,3039 0,01305
1 - 4 - 11,707 0,0006 0,57570 2,2265 0,05014
1 - 5 - 11,932 0,0001 0,23129 0,8572 0,40689
1 - 6 - 11,930 0,0001 0,45188 2,0262 0,05976
Positive and negative controls 
Treatment S9  Plate counts Mean S. E.
Untreated  -  133 140 137 137 2.0
Sodium Azide 1 µg/pl  - 1075 1069 1003 1049 23.1
* = thinning of the background lawn

TETRAMETHRIN: REVERSE MUTATION INS. typhimurium

TABLE 13 - Experiment II      - Plate incorporation method -

STUDY NO.: 9324

SOLVENT: DMSO

Strain: TA100 Titre: 224
Dose-level
[µg/pl]
With metabolic activation
Plate counts Mean S.E.
Untreated 138 132 138 136 2,0
0,00 143 134 145 141 3,4
313 199 212 200 204 4,2
625 254 251 225 243 9,2
1250 262 262 234 253 9,3
2500 248 247 261 252 4,5
5000 253 235 263 250 8,2
Regression analysis:
Points S9 Intercept Slope Corr. coeff. t P-value
1 - 3 + 12,039 0,0060 0,96884 10,3485 0,00002
1 - 4 + 12,763 0,0030 0,85286 5,1652 0,00042
1 - 5 + 13,531 0,0012 0,70021 3,5363 0,00365
1 - 6 + 14,093 0,0005 0,56022 2,7053 0,01560
Positive and negative controls 
Treatment S9 Plate counts Mean s.e.
DMSO 100 µl/p1 + 143 134 145 141 3,4
2-Aminoanthracene 1 µl/p1 + 1246 1243 1222 1237 8
Conclusions:
Although increases in revertant numbers were observed in the presence of S9 with TA100 tester strain (the dose response was not reproduced in a second experiment), the criteria for a clear mutagenic effect were not fully met and the outcome for the test item TETRAMETHRIN was regarded as inconclusive. All other tester strains assessed (TA1535, TA1537, TA98 and TA102) were clearly negative (with and without S9).
Executive summary:

The test item TETRAMETHRIN was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic organism Salmonella typhimurium.

The five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and beta naphthoflavone.

In the toxicity test, the test item was assayed at a maximum dose-level of 5000 µg/plate and at four lower dose-levels spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No signs of toxicity were observed at any dose-level tested, in any tester strain, in the absence or presence of S9 metabolic activation.

Two main experiments were performed.

In Main Assay I, using the plate incorporation method, the test item was assayed at a maximum dose-level of 5000 µg/plate and at four lower dose-levels, separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate. Increases in revertant numbers, which were at least two-fold greater than the control value, were observed at the higher dose-levels with TA100 tester strain in the presence of S9 metabolism.

On the basis of these results, in Main Assay II the same dose-levels used in Main Assay I were employed. A pre-incubation step was included in all treatments with the exception of testing TA100 in the presence of S9 metabolism. With this tester strain the test item was assayed (in the presence of S9 metabolism) under the same experimental conditions employed in Main Assay I. Increases in revertant colonies were again observed in TA100 in the presence of metabolic activation, but these increases did not reach twice the control value.

Precipitation of the test item was observed at 5000 and 2500 µg/plate.

It is concluded that, although increases in revertant numbers were observed in the presence of S9 with TA100 tester strain, the results obtained preclude making a definite judgement about the activity of the test item TETRAMETHRIN.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: EEC Council Directive 2000/32, Annex 4A
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
batch TT-SF-01-001
Target gene:
n/a
Clastogenic activity of TETRAMETHRIN in Chinese hamster ovary cells has been evaluated
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary cells were obtained from Dr. A.T. Natarajan (State University of Leiden). This cell line derives from the CHO isolate originally described by Kao and Puck (1968).
The karyotype, generation time and plating efficiency have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
Permanent stocks of CHO cells are stored in liquid nitrogen, and subcultures are prepared from these stocks for experimental use. Cultures are grown in Ham's F I 0 medium supplemented with 15% Newborn Calf serum. All incubations are at 37°C in a 5% carbon dioxide atmosphere (100% humidity nominal). Culture flasks arc labelled during subculturing and experimental use with their identity and appropriate codices.
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate
Test concentrations with justification for top dose:
The first assay employed dose-levels of 200, 100, 50.0, 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 µg/ml both in the absence and presence of S9 metabolism, while the same dose range, with the exception of 0.781 µg/ml, was used in the second assay.
Dose-levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of cell counts at the time of harvesting.
The treatment-levels selected for scoring were the following:
Experiment S9 Treatment time (hours) Harvest time (hours) Dose-level (µg/ml)
1 - 3 20 100. 50.0 and 25.0
+ 3 20 200, 100 and 50.0
2 - 21 21 50.0, 25.0 and 12.5
Vehicle / solvent:
Dimethylsulphoxide (DMSO) batches no 417035/1 41501 and 421649/1 obtained from Fluka.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
first experiment, 3 h treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
first experiment, 3 h treatment
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
second experiment, 21 h treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations excluding gaps, was observed at any dose-level selected for the scoring in the presence or absence of S9 metabolism.
For the first experiment, statistically significant increases in the incidences of cells bearing aberrations including gaps were observed at the intermediate dose-level selected for scoring both in the absence and presence of S9 metabolism (100 and 50.0 µg/ml respectively).
A statistically significant increase in the incidence of cells bearing aberrations including gaps was also observed for the second experiment at the highest dose-level selected for scoring (50.0 µg/ml)
Statistically significant increases in the incidences of aberrant cells (both including and excluding gaps) compared with the relevant control values, were observed in the cultures treated with the positive controls Cyclophosphamide and Mitomycin-C, indicating the correct functioning of the assay system.
Conclusions:
It must be concluded that the test item does not induce chromosomal aberrations in Chinese hamster ovary cells in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item TETRAMETHRIN was assayed for the ability to cause chromosomal damage in Chinese hamster ovary cells, following in vitro treatment in the absence and presence of S9 metabolic activation.

Two assays for chromosomal damage were performed. The first assay employed dose-levels of 200, 100, 50.0, 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 µg/ml both in the absence and presence of S9 metabolism, while the same dose range, with the exception of 0.781 µg/ml, was used in the second assay.

Solutions of the test item were prepared in dimethylsulphoxide (DMSO).

In the first assay, both in the absence and presence of S9, the cells were treated for 3 hours and the harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used. As negative results were obtained, a second assay in the absence of S9 metabolism was performed using a continuous treatment until harvest at 21 hours.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

Dose-levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of cell counts at the time of harvesting.

The treatment-levels selected for scoring were the following:

Experiment

S9

Treatment time
(hours)

Harvest time
(hours)

Dose-level
(µg/ml)

1

-

+

3

3

20

200, 100 and 50.0
100. 50.0 and 25.0

2

-

21

21

50.0, 25.0 and 12.5

One hundred metaphase spreads were scored for chromosomal aberrations from each culture treated with the test item. For control cultures treated with Mitomycin-C and Cyclophosphamide, where more than 50% of cells presented chromosome aberrations (excluding gaps), scoring was terminated at 50 metaphases.

Following treatment with TETRAMETHRIN, no statistically significant increase in the incidence of cells bearing aberrations (excluding gaps), compared with the relevant control values, was observed at any dose-level selected for the scoring in the absence or presence of S9 metabolism.

For the first experiment, a statistically significant increase in the incidence of cells bearing aberrations including gaps was observed at the intermediate dose-level selected for scoring (100 µg/ml) in the absence of S9 metabolism. A statistically significant increase in the incidence of cells bearing aberrations including gaps was also observed at the intermediate dose-level selected for scoring (50 µg/ml) in the presence of S9 metabolism.

For the second experiment, a statistically significant increase in the incidence of cells bearing aberrations including gaps was observed at the high dose-level (50.0 µg/ml) selected for scoring in the absence of S9 metabolism.Statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatments with the positive controls Cyclophosphamide and Mitomycin-C, indicating the correct functioning of the test system.

It is concluded that TETRAMETHRIN does not induce chromosomal aberrations in Chinese hamster ovary cells after in vitro treatment in the presence or absence of metabolic activation, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
other: EEC Council Directive 2000/32 Annex 4E
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
5-trifluorothymidine
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fractions from male Sprague-Dawley rats pre-treated with phenobarbital and beta-naphthoflavone, see Table 1
Test concentrations with justification for top dose:
After performing a solubility and cytotoxicity test, two independent assays for mutation to trifluorothymidine resistance were performed using dose-levels described in the following table

Assay No.: S9 Treatment Time (hours) Dose-level (µg/ml)
1 - 3 40.0, 30.0, 25.0, 12.5, 6.25, 3.13 and 1.56
1 + 3 25.0, 12.5, 6.25, 3.13 and 1.56
2 - 24 75.0, 50.0, 25.0, 12.5, 6.25 and 3.13
2 + 3 75.0, 50.0, 37.5, 25.0, 16.7, 11.1 and 7.41
Vehicle / solvent:
Dimethylsulphoxide (DMSO) obtained from Fluka AG, Buchs, Switzerland (batches no.: 417035/1 41501 and 421649/1 13001)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
L5178Y TK +/- (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
Permanent stocks of the L5178Y TK +/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
The mutation assay was conducted in 96-well plates.
Evaluation criteria:
In the original study report, a chi² analysis was used to identify significant increases in mutation frequency. A linear trend analysis was used to identify a dose response. Upon re-evaluation, this was extended using the 95%-CI of the historical control range and the Global Evaluation Factor approach, both laid down in OECD TG 490.
Statistics:
chi² analysis and test for linear trend
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
first experiment-3 h treatment
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
first experiment- 3 h treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
second experiment- 3 h treatment
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
did not exceed the GEF
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
second experiment- 24 h treatment
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
GEF exceeded only for high conc. (50 µg/mlL)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
25% RS
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Results of the solubility test
The test item was found to be soluble in DMSO at a maximum concentration of 300 mg/ml. A dilution series in DMSO was prepared and aliquots were added to RPMI complete medium (10%) in the ratio 1 : 100. The addition of stock dilutions between 300 and 37.5 mg/ml generated precipitation, while an aliquot at 18.7 mg/ml generated a clear solution. On the basis of this result a concentration of 400 µg/ml was selected as the highest dose-level to be used in the cytotoxicity test.

Result of the Cytotoxicity test

Treatment time - 3 hours
In the cytotoxicity test, the test item was assayed at a maximum concentration of 400 µg/ml and a wide range of lower dose-levels: 200, 100, 50.0, 25.0, 12.5, 6.25, 3.13 and 1.56 µg/ml. At the end of the treatment incubation period, precipitation was observed at the two higher concentrations tested.
Following treatment in the absence of S9 metabolic activation, no cells survived at the four higher concentrations. Moderate toxicity was observed at 25.0 µg/ml, while slight or no toxicity was observed at the remaining dose-levels. In the presence of S9 metabolic activation, no cells survived at the four higher concentrations, while dose-dependent toxicity was observed at the remaining concentrations reducing survival to 13% of the negative control value at 25.0 µg/ml.

Treatment time - 24 hours
Concentrations tested were: 400, 200, 100, 50.0, 25.0, 12.5, 6.25, 3.13 and 1.56 µg/ml.
Following treatment, no cells survived at the three higher concentrations, while slight or no toxicity was observed at the remaining dose-levels.

Table 4          Summary Table of Mutation assays results with and without S9

 

Experiment 1

Dose-level

(μg/mL)

WITHOUT S9

(Treatment time: 3 hours)

Dose-level

(μg/mL)

WITH S9

(Treatment time: 3 hours)

% RS

MF°

P

% RS

MF°

P

Hist. Control#

 

30.4-162.2

 

 

 

34.4-170.0

 

0.00

100

51.01

-

0.00

100

60.14

-

1.56

70

45.54

NS

1.56

100

57.79

NS

3.13

73

52.61

NS

3.13

100

60.39

NS

6.25

99

59.55

NS

6.25

87

52.07

NS

12.5

84

70.31

NS

12.5

87

63.40

NS

25.0

74

47.96

NS

25.0

78

60.86

NS

30.0

76

60.61

NS

Control + GEF$

 

186.1

 

40.0

47

66.79

NS

 

 

 

 

Control + GEF$

 

177.0

 

 

 

 

 

 

Linear trend

NS

 

Linear trend

NS

 

Experiment 2

Dose-level

(μg/mL)

WITHOUT S9

(Treatment time: 24 hours)

Dose-level

(μg/mL)

WITH S9

(Treatment time: 3 hours)

% RS

MF°

P

% RS

MF°

P

Hist. Control#

 

13.5-180.5

 

 

 

34.4-170.0

 

0.00

100

80.71

-

0.00

100

106.3

-

3.13

106

86.95

NS

7.41

109

157.7

NS

6.25

115

108.0

NS

11.1

101

97.04

NS

12.5

104

99.61

NS

16.7

98

103.2

NS

25.0

91

122.4

NS

25.0

99

97.12

NS

Control + GEF$

 

206.7

 

37.5

87

147.3

NS

50.0

25

370.8

NS

50.0

81

178.7

<5%

75.0

0

*

<1%

75.0

57

197.5

<1%

 

 

 

 

Control + GEF$

 

232.3

 

 

Linear trend

<0.1%

 

Linear trend

<0.1%

 

° = Figures displayed are mutation frequencies (MF) per million surviving cells

RS = relative survival.

MF = Mutant Frequency

#95% CI from the performing laboratory, calculated from the SD (× 1.96) given in the study report

$GEF = Global Evaluation Factor for microwell version of MLA (126 E-06, OECD TG 490, 28 July 2015)

* = Not plated for viability/5-TFT resistance

 

 

Table 5          Small and large colony mutant frequencies

Experiment 1

Dose-level

(μg/mL)

S9

Mutant frequency

Proportion small colony mutants

Small colony

Large colony

0.00

-

10.0

39.7

0.20

MMS 10.0

-

77.3

178

0.30

 

 

 

 

 

0.00

+

11.9

46.9

0.20

B(a)P 2.00

+

85.1

315

0.21

 

Experiment 2

Dose-level

(μg/mL)

S9

Mutant frequency

Proportion small colony mutants

Small colony

Large colony

0.00

-

17.1

60.6

0.22

50.0

-

120

193

0.38

MMS 5.00

-

174

327

0.35

 

 

 

 

 

0.00

+

25.5

75.9

0.25

50.0

+

51.7

112

0.32

75.0

+

69.8

109

0.39

B(a)P 2.00

+

340

459

0.43

 

° = Figures displayed are mutation frequencies per 106viable cells

Conclusions:
An increase in mutant frequency above the Global Evaluation Factor was observed after continuous 24-h treatment with TETRAMETHRIN in the absence of S9 metabolism. However, this increase occurred in the presence of significant cytotoxicity (25% RS). On the basis of this results, the criteria for a clear mutagenic effect were not met and the outcome was regarded as equivocal.
Executive summary:

The test item TETRAMETHRIN was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment (in the absence and presence of S9 metabolic activation) using a fluctuation method in 96-well plates.

Test item solutions were prepared using dimethylsulphoxide.

A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose-level of 400 µg/ml and a wide range of lower dose-levels: 200, 100, 50.0, 25.0, 12.5, 6.25, 3.13 and 1.56 µg/ml. Upon addition of the test item to the cultures precipitation was observed at the two higher dose-levels. At the end of the 3 hour treatment incubation period, precipitation was present at the same dose-levels, while after 24 hours no precipitation was observed. Using a short treatment time (3 hours) in the absence of S9 metabolic activation, no cells survived at the four higher concentrations. Moderate toxicity was observed at 25.0 µg/ml, while slight or no toxicity was observed at the remaining dose-levels. In the presence of S9 metabolic activation, no cells survived at the four higher concentrations, while dose-dependent toxicity was observed at the remaining concentrations reducing survival to 13% of the negative control value at 25.0 µg/ml. Using a long treatment time (24 hours), no cells survived at the three higher concentrations, while slight or no toxicity was observed at the remaining dose-levels

Two independent assays for mutation at the TK locus were performed using dose-levels described in the following table:

Assay No.:

S9

Treatment
Time (hours)

Dose-level (µg/mL)

1

 

3

40.0, 30.0, 25.0, 12.5, 6.25, 3.13 and 1.56

1

+

3

25.0, 12.5, 6.25, 3.13 and 1.56

2

 

24

75.0, 50.0, 25.0, 12.5, 6.25 and 3.13

2

+

3

75.0, 50.0, 37.5, 25.0, 16.7, 11.1 and 7.41

 

A statistically significant increase in mutant frequency was observed following treatment with the test item in the absence of S9 metabolism when the long treatment time was used. This increase was observed only at the highest concentration and was not reproduced when a 3-hour treatment time was used. In the presence of S9 metabolic activation, no increases in mutant frequency were observed in the first experiment, at any dose-levels tested. In the second experiment, using a modified dose-range to obtain an adequate level of toxicity, a linear trend was indicated and slight, but statistically significant increases in mutant frequency were observed at the two higher concentrations. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the negative control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.  An increase in mutant frequency above the Global Evaluation Factor was observed after continuous 24-h treatment with TETRAMETHRIN in the absence of S9 metabolism. However, this increase occurred in the presence of significant cytotoxicity (25% RS). On the basis of this results, the criteria for a clear mutagenic effect were not met and the outcome was regarded as equivocal.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1984
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Qualifier:
according to
Guideline:
other: E.coli DNA repair test
GLP compliance:
no
Type of assay:
other: in vitro DNA damage and/or repair study
Specific details on test material used for the study:
Purity > 92%
Species / strain / cell type:
E. coli, other: W3110/polA+
Species / strain / cell type:
E. coli, other: p3478/polA-
Test concentrations with justification for top dose:
Technical tetramethrin was tested at 6 doses: 100 µg, 333 µg, 1000 µg, 3333 µg, 10000 µg and 33333 µg per plate.
Vehicle / solvent:
Technical tetramethrin (Batch 13/83) was dissolved in dimethylsulphoxide to give a stock solution of 333 mg/mL, from which half-log dilutions were prepared.
Untreated negative controls:
yes
Remarks:
Chloramphenicol
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, caffeine
Evaluation criteria:
For a compound to be judged as a modifier of cellular DNA it must preferentially inhibit E. coli p3478/polA- by providing a 4 mm differential in the diameter of the zone of growth inhibition.
If growth inhibition is not observed with either strain, then the experiment is judged to have given inconclusive results.
Species / strain:
E. coli, other: W3110/polA+
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli, other: p3478/polA-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The response obtained with methyl methanesulphonate (MMS) was considered inadequate in the first test on technical tetramethrin. A retest was therefore performed using MMS, AF-2 and caffeine, and on this occasion these substances gave the expected response by preferentially killing E. coli p3478/polA-. The non-specific control, chloramphenicol, killed both strains of E. coli to an equal extent. Dimethylsulphoxide, the solvent for technical tetramethrin, was not toxic to either strain.

The results of the first and second test on technical tetramethrin (Batch 13/83) show that technical tetramethrin was not toxic to either strain of bacteria in the presence or absence of S-9 mix when tested to a highest dose of 33 mg per plate. Precipitation of test substances was noted from a lowest dose of 1.0 mg per plate.

Conclusions:
No toxicity due to technical tetramethrin was noted in either strain. The results of this study, therefore, suggest that technical tetramethrin does not damage bacterial DNA. However, since no toxic response was observed at all, it is concluded in addition that technical tetramethrin is innocuous to E. coli at the highest attainable concentration, but the bioavailability of the compound may be low in the test system.
Executive summary:

Technical tetramethrin (Batch No. 13/83) was tested for its ability to cause differential growth inhibition in Escherichia coli H3110/polA+ and p3478/polA-. The test was conducted on agar plates both with and without rat liver foreign compound metabolising enzymes and the co-factors necessary for mono-oxygenase activity. It is considered to be a test for DNA damage repairable by enzyme systems involving DNA polymerase.

Technical tetramethrin was tested at 6 doses: 100 µg, 333 µg, 1000 µg, 3333 µg, 10000 µg and 33333 µg per plate.

Technical tetramethrin was not toxic to either strain of bacteria in the presence or absence of S-9 mix and therefore, no evidence was found to suggest it might damage bacterial DNA.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A mammalian erythrocyte micronucleus test has been performed on test item tetramethrin according to OECD guideline no. 474. The test item tetramethrin was not mutagenic in this micronucleus test in Swiss albino mice at the tested limit dose of 2000 mg/kg under the testing conditions adopted.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
Batch No.: TM1027
Species:
mouse
Strain:
other: Swiss albino (HsdOla : MF1 strain)
Details on species / strain selection:
Source: Toxicology department
Advinus Therapeutics Private Limited
Bangalore —560 058
India
Sex:
male/female
Details on test animals and environmental conditions:
Age at the start of acclimatisation: 11 weeks
Acclimatisation: Five days under experimental conditions after veterinary examination.
Grouping: Mice were assigned to 3 study groups by body weight stratification during acclimatisation.
Body weight range after grouping: Males : 27.69 — 37.10 g Females : 24.60 — 29.10 g.
Identification: At the start of acclimatisation, mice were identified by serial numbers and crystal violet body marking. After grouping, they were identified by turmeric body marking, cage cards and permanent accession numbers.
Route of administration:
oral: gavage
Vehicle:
Carboxymethyl cellulose (sodium salt)
Details: 0.5% aqueous carboxymethyl cellulose (CMC) with Tween 80 (1 mL/L)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

TEST ITEM PREPARATION FOR ORAL GAVAGING: Just prior to the treatment, a precisely weighed aliquot of 4.0 g of the finely ground test item (weighed using an electronic balance) was suspended in 0.5% aqueous CMC with Tween 80 (1mI/L) and the final volume was made up to 20 ml to obtain the nominal concentration of 200 mg of the test item per ml of the suspension. The homogeneity of the test item suspension was maintained by constant stirring using a magnetic stirrer.

PREPARATION OF POSITIVE CONTROL FOR ORAL GAVAGING: Before each treatment, 40 mg of cyclophosphamide (monohydrate) was dissolved in 10 ml of distilled water in a clean glass beaker to obtain a concentration of 4 mg/ml of the solution.
Duration of treatment / exposure:
48 h
Frequency of treatment:
The test item, the positive control and the vehicle control were administered orally as gavage twice at 24 hour intervals to each group of animals.
Post exposure period:
The animals were sacrificed 23 to 24 hours after the second treatment.
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
10 animals (5 males + 5 females)/group
3 groups have been tested: 1 test item concentration, 1 positive control and 1 vehicle control
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control group has been exposed to 40 mg/kg of cyclophosphamide (monohydrate).
Tissues and cell types examined:
Erythrocytes from bone marrow
Details of tissue and slide preparation:
Terminal Sacrifice
The treated mice were sacrificed 23 - 24 hours after the second treatment for sampling of femur bone marrow cells. All mice sacrificed at term were subjected to gross pathological observations. The mice were killed by cervical dislocation and the femora from both sides were removed after clearing the musculature. Femur heads were trimmed to expose the marrow canals, the bone marrow was flushed with 0.9% sodium chloride and collected in a centrifuge tube.
Bone Marrow Smear Preparation
The cell suspensions were centrifuged at 2500 - 2750 rpm for 10 minutes and supernatants discarded. Approximately 10 microlitre of the cell suspension was spread evenly on a glass slide and air-dried. The slide was etched with the study number, mouse accession number, slide number and sex immediately thereafter. The smears were fixed in methanol for about 30 minutes. 4 to 5 slides were prepared for each animal.
Staining
Slides were stained by a combination of May-Gruenwald and Giemsa stain in succession. The slides were blow-dried, immersed in xylene and cover slips mounted with DPX. The slides were then coded after which blind evaluation was carried out.




Evaluation criteria:
A minimum of 2000 polychromatic erythrocytes (PCE) was scored from each animal for the incidence of PCE with micronuclei. The proportion of immature erythrocytes among total RBC (number of PCE divided by number of total erythrocytes) was determined for each animal by counting 295 to 556 erythrocytes per animal.
From these observations, the following were derived for each animal:
·       Total RBC/erythrocytes scored
·       No. of PCE differentiated
·       No. and percentage of PCE with micronuclei
·       Mean and SD of PCE with micronuclei
·       Ratio of PCE : total RBC
After completing the evaluation of all the slides, the slides were verified or cross-checked by another in-house cytogeneticist. This included verification of all the micronucleated PCE in the vehicle control and the treatment group and 10% of the micronucleated PCE in the positive control group. The micronucleated PCE to be verified were chosen using the random numbers generation method as per the in-house developed and validated randomisation program.
Statistics:
For intragroup comparison, the body weights on day 2 and at sacrifice were compared with the initial body weight, and the body weight at sacrifice was also compared with the body weight on day 2 by paired 't' test. (Snedecor and Cochran, 1980).
For intergroup comparison, the net body weight change was analysed by Bartlett's test for homogeneity of intragroup variances followed by ANOVA and Dunnett's test. (Sokal and Rohlf, 1981).
The data of the treatment group and the positive control group was compared with the vehicle control by Bartlett's test followed by ANOVA and Dunnett's test (Gomez and Gomez, 1984) for the percentage of PCE with micronuclei and the proportion of PCE among total RBC.
The above statistical analyses were carried out using in-house developed and validated software.
All analyses and comparisons were evaluated at 5% (p <0.05) level
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Vehicle Control Group
There were no incidences of micronucleated PCE in males in the vehicle control group. The incidences of micronucleated PCE in females and PCE : total RBC ratio in males, females and combined sex in the concurrent vehicle control group was within the historical data range.

Treatment Group
The incidences of micronucleated PCE and the PCE : total RBC ratio in the limit dose group in males, females and combined sex were comparable to the vehicle control values.

Positive Control Group
Cyclophosphamide (monohydrate) significantly increased the percentage of micronucleated PCE in combined sex and significantly altered (reduced) the PCE : total RBC ratio in males, females and combined sex. The percentage of micronucleated PCE and PCE: Total RBC ratio was within the positive control historical data range in males, females and combined sex.


CLINICAL SIGNS, MORTALITY AND NECROPSY FINDINGS

There were no clinical signs, mortality or necropsy findings in the tested dose groups in males, females and combined sex.

BODY WEIGHT

In the limit dose group, the intra group comparison of the body weights showed a statistically significant increase:

a.       In sacrifice body weight as compared to the initial body weight in combined sex.

b.       In sacrifice body weight as compared to day 2 body weight in females and combined sex.

The intergroup comparison of the net body weight change showed:

a.       A statistically significant increase in combined sex in the limit dose group.

b.       A decrease in body weights in females and combined sex in the vehicle control group.

As the treatment increased the body weights of mice as compared to the concurrent control group, it was inferred that treatment did not affect the body weights of mice.

In the positive control group, the intragroup comparison of body weights as compared to the initial body weights showed a statistically significant decrease:

a.       In day 2 body weights in females and combined sex

b.       In sacrifice body weights in combined sex.

The intergroup comparison of net body weight change showed a decrease in body weights in males, females and combined sex as compared to the vehicle control group

Conclusions:
The test item tetramethrin was not mutagenic in this micronucleus test in Swiss albino mice at the tested limit dose of 2000 mg/kg under the testing conditions adopted.


Executive summary:

Tetramethrin was tested for its potential to induce cytogenetic damage in bone marrow cells of Swiss albino mice by a micronucleus test. The test item was administered twice at an interval of 24 hours by oral gavage to a group of Swiss albino mice at the limit dose of 2000 mg/kg body weight at the dosage volume of 10 ml/kg. A concurrent vehicle control group {0.5% Carboxy methyl cellulose with Tween 80 1 mI/L} and a concurrent positive control group {cyclophosphamide (monohydrate)} were also included. The mice were sacrificed 23 - 24 hours after the second treatment. From each animal, a minimum of 2000 polychromatic erythrocytes (PCE) were scored for the incidence of micronucleated PCE. The ratio of PCE : total RBC was determined by counting 295 - 556 RBC per animal.

The results are summarized as follows:

Tetramethrin at the tested limit dose of 2000 mg/kg body weight did not affect the body weights of mice and did not cause clinical signs, mortality or gross lesions.

There were no incidences of micronucleated PCE in males in the vehicle control group. The incidences of micronucleated PCE in females and PCE : total RBC ratio in males, females and combined sex in the concurrent vehicle control group was within the historical data range.

The incidences of micronucleated PCE and the PCE : total RBC ratio in the limit dose group in males, females and combined sex was comparable to the vehicle control values.

Cyclophosphamide (monohydrate) significantly increased the percentage of micronucleated PCE in combined sex and significantly altered (reduced) the PCE : total RBC ratio in males, females and combined sex. The percentage of micronucleated PCE and PCE: Total RBC ratio was within the positive control historical data range in males, females and combined sex.

This result indicated the effectiveness of the methodology adopted and that the test system used was sensitive to a known mutagen.

The test item tetramethrin was not mutagenic in this micronucleus test in Swiss albino mice at the tested limit dose of 2000 mg/kg body weight under the conditions adopted.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on an overall weight of evidence by four in vitro and one in vivo mutagenicity studies, it is concluded that tetramethrin does not require classification for mutagenicity according to CLP (Regulation EC No. 1272/2008).