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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October 2018 - 17 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical BV (The Netherlands); Lot No. 1803-1
- Expiration date of the lot/batch: 2020-01-15
- Purity test date: 2018-09-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15-25°C, protected from light under nitrogen
- Stability under test conditions: no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparations of test article were employed
- Solubility and stability of the test substance in the solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test article stock solutions were prepared by formulating Kalama® Benzaldehyde FCC grade under subdued lighting in DMSO, with the aid of vortex mixing, to give
the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 4 hours of initial formulation.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : colourless liquid

Method

Species / strain
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood from two healthy, non-smoking Female volunteers from a panel of donors at Covance was used for each experiment.
- Suitability of cells: The use of human peripheral blood lymphocytes is recommended because the cells are only used in short-term culture and maintain a stable karyotype (Evans & O’Riordan, 1975).
- Cell cycle length, doubling time or proliferation index: Cell cycle length: 13 ± 2 hour
- Sex, age and number of blood donors if applicable: Blood from two healthy, non-smoking Female volunteers (Range-Finder: 34, 30 years; Micronucleus Experiment: 23, 34 Years)
- Whether whole blood or separated lymphocytes were used if applicable: human lymphocyte cultures prepared from the pooled blood of two female donors
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Cytotoxicity Range-Finder Experiment:
3+21 Hour Treatment (-S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL
3+21 Hour Treatment (+S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL
24+24 Hour Treatments (-S9): 3.853, 6.422, 10.70, 17.84, 29.73, 49.55, 82.58, 137.6, 229.4, 382.3, 637.2, 1062 µg/mL

A maximum concentration of 1062 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a maximum concentration equivalent to 10 mM (a suitable maximum concentration for in vitro genetic toxicology assays of this type), based on the test article molecular weight of 106.121. No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1062 µg/mL), compared to the concurrent vehicle controls. The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for the Micronucleus Experiment.

Micronucleus Experiment:
3+21 Hour Treatments (-S9): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 950, 1062 µg/mL
3+21 Hour Treatments (+S9): 100, 200, 400, 500, 600, 650, 700, 750, 800, 850, 950, 1062 µg/mL
24+24 Hour Treatments (-S9): 50, 100, 150, 200, 250, 300, 350, 400, 450, 600 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Kalama® Benzaldehyde FCC grade was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 119.5 mg/mL.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Vinblastine (VIN): Treatment Regime: -S9: 24+24 (concentration: 0.04 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: approximately 48 hours
- Exposure duration: 3 (±S9) or 24 hours (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): -S9 (3 hour treatment): 72 hours; -S9 (24 hour treatment): 96 hours; + S9 (3 hour treatment): 72 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (Cyto-B (formulated in DMSO) added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline (PBS), pH 6.8

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

NUMBER OF CELLS EVALUATED: A minimum ofone thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. For the 24 hour treatment in the absence of S9, an additional 1000 binucleate cells from each culture (therefore 4000 per concentration) were analysed from the test article concentrations selected for analysis.


CRITERIA FOR MICRONUCLEUS IDENTIFICATION: A micronucleus was only recorded if it met the following criteria:

1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Relative index (RI)
- Any supplementary information relevant to cytotoxicity: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined. RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formula as follows:

RI = (number binucleate cells + 2 (number multinucleate cells) / total number of cells in treated cultures)

Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:

Relative RI (%) = (RI of treated cultures / RI of vehicle controls) x 100

Cytotoxicity (%) is expressed as (100 – Relative RI)

A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.
Rationale for test conditions:
Please see 'Any other information on materials and methods incl. tables' for information on Rationale for Test Conditions.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:

1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed

2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed

3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations (Thybaud et al., 2007).
Statistics:
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.

The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).

The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p ≤0.05 were accepted as significant.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No marked changes in osmolality or pH were observed at the highest concentration tested in the Range-Finder (1062 µg/mL), compared to the concurrent vehicle controls (individual data not reported).

The proportion of micronucleated binucleate (MNBN) cells in negative (vehicle) control cultures fell within (or very close to) the 95th percentile of the current observed historical vehicle control (normal) ranges. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was accepted as valid.


Validity of Study
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures for the 3+21 hour and 24+24 hour treatments in the absence of S9. Statistically significant heterogeneity (p≤0.01) was observed for the 3+21 hour treatment in the presence of S9, primarily due to large differences in MNBN cell frequencies between replicates for the vehicle control and the lowest test article concentration analysed (400 µg/mL). However, none of the MNBN cell frequency values exceeded the normal range under this treatment condition, therefore the observed heterogeneity did not affect the interpretation of the data

2. The frequency of MNBN cells in vehicle controls fell within the normal ranges with the exception of two of the four vehicle control cultures analysed for the 24+24 hour treatment in the absence of S9, one of which fell within the observed range. However, the mean vehicle control MNBN cell frequency was within the normal range and the data were considered acceptable.

3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range.

4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.

5. The maximum concentration analysed under each treatment condition met the criteria specified.

Any other information on results incl. tables

Table 4. Range-Finder: -S9: Results of the 3+21 Hour Treatments

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity

Based on RI (%)

Vehicle

A

64

125

11

200

0.74

 

B

52

133

15

200

0.82

 

Total

116

258

26

400

0.78

-

3.853

A

NSc

-

-

-

-

-

6.422

A

NSc

-

-

-

-

-

10.70

A

NSc

-

-

-

-

-

17.84

A

NSc

-

-

-

-

-

29.73

A

42

148

10

200

0.84

0

49.55

A

58

139

3

200

0.73

6

82.58

A

49

140

11

200

0.81

0

137.6

A

54

135

11

200

0.79

0

229.4

A

52

138

10

200

0.79

0

382.3

A

75

123

2

200

0.64

18

637.2

A

96

104

0

200

0.52

33P

1062

A

183

17

0

200

0.09

89P

P = Precipitation observed at treatment

NSc = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

Table 5. Range-Finder: +S9: Results of the 3+21 Hour Treatments

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity

Based on RI (%)

Vehicle

A

50

139

11

200

0.81

 

B

56

127

17

200

0.81

 

Total

106

266

28

400

0.81

-

3.853

A

NSc

-

-

-

-

-

6.422

A

NSc

-

-

-

-

-

10.70

A

NSc

-

-

-

-

-

17.84

A

64

122

14

200

0.75

7

29.73

A

55

133

12

200

0.79

2

49.55

A

58

137

5

200

0.74

9

82.58

A

45

138

17

200

0.86

0

137.6

A

59

132

9

200

0.75

7

229.4

A

57

138

5

200

0.74

8

382.3

A

78

118

4

200

0.63

22

637.2

A

96

102

2

200

0.53

34P

1062

A

177

23

0

200

0.12

86P

P = Precipitation observed at treatment

NSc = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

Table 6. Range-Finder: +S9: Results of the 24+24 Hour Treatments

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity

Based on RI (%)

Vehicle

A

28

146

26

200

0.99

 

B

39

142

19

200

0.90

 

Total

67

288

45

400

0.95

-

3.853

A

NSc

-

-

-

-

-

6.422

A

NSc

-

-

-

-

-

10.70

A

NSc

-

-

-

-

-

17.84

A

31

148

21

200

0.95

0

29.73

A

24

152

24

200

1.00

0

49.55

A

23

155

22

200

1.00

0

82.58

A

48

144

8

200

0.80

15

137.6

A

80

118

2

200

0.61

35

229.4

A

124

74

2

200

0.39

59

382.3

A

119

74

7

200

0.55

53

637.2

A

194

6

0

200

0.03

97P

1062

A

199

1

0

200

0.01

99P

P = Precipitation observed at treatment

NSc = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

Table 7. Micronucleus Experiment: -S9: Results of the 3+21 Hour Treatments

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity

Based on RI (%)

Vehicle

A

112

360

28

500

0.83

 

B

127

347

26

500

0.80

 

C

85

380

35

500

0.90

 

D

88

378

34

500

0.89

 

Total

412

1465

123

2000

0.86

-

100.0

A

134

344

22

500

0.78

 

B

114

365

21

500

0.81

 

Total

248

709

43

1000

0.80

7

200.0

A

143

328

29

500

0.77

 

B

160

315

25

500

0.73

 

Total

303

643

54

1000

0.75

12

400.0

A

140

348

12

500

0.74

 

B

152

337

11

500

0.72

 

Total

292

685

23

1000

0.73

15 #

500.0

A

183

311

6

500

0.65

 

B

170

325

5

500

0.67

 

Total

353

636

11

1000

0.66

23

600.0

A

213

286

1

500

0.58

 

B

244

255

1

500

0.51

 

Total

457

541

2

1000

0.55

36 #

650.0

A

246

249

5

500

0.52

 

B

237

261

2

500

0.53

 

Total

483

510

7

1000

0.52

39

700.0

A

225

271

4

500

0.56

 

B

279

219

2

500

0.45

 

Total

504

490

6

1000

0.50

41

750.0

A

265

234

1

500

0.47

 

B

285

213

2

500

0.43

 

Total

550

447

3

1000

0.45

47

800.0

A

305

193

2

500

0.39

 

B

294

206

0

500

0.41

 

Total

599

399

2

1000

0.40

53 #

850.0

A

308

191

1

500

0.39

 

B

303

194

3

500

0.40

 

Total

611

385

4

1000

0.39

54

950.0

A

348

151

1

500

0.31

 

B

381

117

2

500

0.24

 

Total

729

268

3

1000

0.27

68 P

1062

A

473

27

0

500

0.05

 

B

456

44

0

500

0.09

 

Total

929

71

0

1000

0.07

92 P

MMC, 0.30 

A

250

249

1

500

0.50

 

B

244

255

1

500

0.51

 

Total

494

504

2

1000

0.51

41 #

P = Precipitation observed at treatment

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

# Highlighted concentrations selected for analysis

 

Table 8. Micronucleus Experiment: +S9: Results of the 3+21 Hour Treatments

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity

Based on RI (%)

Vehicle

A

132

341

27

500

0.79

 

B

117

342

41

500

0.85

 

C

115

369

16

500

0.80

 

D

110

366

24

500

0.83

 

Total

474

1418

108

2000

0.82

-

100.0

A

144

343

13

500

0.74

 

B

106

366

28

500

0.84

 

Total

250

709

41

1000

0.79

3

200.0

A

124

364

12

500

0.78

 

B

103

379

18

500

0.83

 

Total

227

743

30

1000

0.80

2

400.0

A

148

343

9

500

0.72

 

B

130

363

7

500

0.75

 

Total

278

706

16

1000

0.74

10 #

500.0

A

204

292

4

500

0.60

 

B

187

310

3

500

0.63

 

Total

391

602

7

1000

0.62

25

600.0

A

250

247

3

500

0.51

 

B

239

259

2

500

0.53

 

Total

489

506

5

1000

0.52

37 #

650.0

A

258

237

5

500

0.49

 

B

255

244

1

500

0.49

 

Total

513

481

6

1000

0.49

40

700.0

A

230

269

1

500

0.54

 

B

274

226

0

500

0.45

 

Total

504

495

1

1000

0.50

39

750.0

A

270

227

3

500

0.47

 

B

270

230

0

500

0.46

 

Total

540

457

3

1000

0.46

43

800.0

A

321

177

2

500

0.36

 

B

303

197

0

500

0.39

 

Total

624

374

2

1000

0.38

54 #

850.0

A

353

145

2

500

0.30

 

B

317

181

2

500

0.37

 

Total

670

326

4

1000

0.33

59

950.0

A

370

128

2

500

0.26

 

B

366

134

0

500

0.27

 

Total

736

262

2

1000

0.27

67 P

1062

A

415

84

1

500

0.17

 

B

403

96

1

500

0.20

 

Total

818

180

2

1000

0.18

77 P

CPA, 3.00

A

181

312

7

500

0.65

 

B

151

342

7

500

0.71

 

Total

332

654

14

1000

0.68

17

CPA, 5.00

A

218

280

2

500

0.57

 

B

235

264

1

500

0.53

 

Total

453

544

3

1000

0.55

 

CPA, 7.00

A

244

255

1

500

0.51

 

B

243

257

0

500

0.51

 

Total

487

512

1

1000

0.51

37 #

P = Precipitation observed at treatment

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

# Highlighted concentrations selected for analysis

 

Table 9. Micronucleus Experiment: -S9: Results of the 24+24 Hour Treatments

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity

Based on RI (%)

Vehicle

A

33

347

120

500

1.17

 

B

41

326

133

500

1.18

 

C

38

328

134

500

1.19

 

D

32

366

102

500

1.14

 

Total

144

1367

489

2000

1.17

 

50.0

A

46

353

101

500

1.11

 

B

53

342

105

500

1.10

 

Total

99

695

206

1000

1.11

6 #

100.0

A

113

363

24

500

0.82

 

B

124

346

30

500

0.81

 

Total

237

709

54

1000

0.82

30 #

150.0

A

151

331

18

500

0.73

 

B

174

320

6

500

0.66

 

Total

325

651

24

1000

0.70

40

200.0

A

264

230

6

500

0.48

 

B

222

269

9

500

0.57

 

Total

486

499

15

1000

0.53

55 #

250.0

A

337

158

5

500

0.34

 

B

266

222

12

500

0.49

 

Total

603

380

17

1000

0.41

65

300.0

A

285

188

27

500

0.65

 

B

312

175

13

500

0.40

 

Total

697

363

40

1000

0.44

62

350.0

A

212

250

38

500

0.65

 

B

262

202

36

500

0.55

 

Total

474

452

74

1000

0.60

49

400.0

A

275

211

14

500

0.48

 

B

291

199

10

500

0.44

 

Total

566

410

24

1000

0.46

61

450.0

A

348

147

5

500

0.31

 

B

363

134

3

500

0.28

 

Total

711

281

8

1000

0.30

75

600.0

A

478

21

1

500

0.05

 

B

478

22

0

500

0.04

 

Total

956

43

1

1000

0.05

96

VIN, 0.04

A

195

225

80

500

0.77

 

B

227

199

74

500

0.69

 

Total

422

424

154

1000

0.73

38 #

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

# Highlighted concentrations selected for analysis

 

Table 10. Summary of Results of the Micronucleus Test

Treatment

Concentration

(µg/mL)

Cytotoxicity

(%)$

Mean

MNBN Cell

Frequency

(%)

Historical

Control

Range (%)#

Statistical

Significance

3+21 hour

-S9

Vehiclea

-

0.60

0.00 to 1.01

-

400.0

15

0.60

NS

600.0

36

0.45

NS

800.0

53

0.50

NS

*MMC, 0.30

41

7.00

p≤0.001

 

3+21 hour +S9

Vehiclea

-

0.50

0.10 to 1.20

-

400.0

10

0.35

NS

600.0

37

0.90

NS

800.0

54

0.80

NS

*CPA, 7.00

37

3.10

p≤0.001

 

24+24 hour

-S9

Vehiclea

-

0.75

0.10 to 0.80

-

50.0

6

0.60

NS

100.0

30

0.40

NS

200.0

55

0.88

NS

*VIN 0.04

38

2.95

p≤0.001

a Vehicle control was DMSO

* Positive control

# 95thpercentile of the observed range

$ Based on replication index

NS Not significant

Applicant's summary and conclusion

Conclusions:
The test material (Kalama® Benzaldehyde FCC grade) did not induce biologically relevant increases in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+24 hours in the absence of S9 under the experimental conditions described.
Executive summary:

In a key OECD Guideline 487 study, the test material (Kalama® Benzaldehyde FCC grade) was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9) from Aroclor 1254-induced rats.

 

The test material was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). The highest concentrations analysed in the Micronucleus Experiment were limited by cytotoxicity under each treatment condition and were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test material concentrations for micronucleus analysis were selected by evaluating the effect of Kalama® Benzaldehyde FCC grade on the replication index (RI). Micronuclei were analysed at three concentrations.

 

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within (or very close to) the 95thpercentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

 

All acceptance criteria were considered met and the study was accepted as valid.

 

Treatment of cells with the test material for 3+21 hours in the absence and presence of S9and for 24+24 hours in the absence of S9 resulted in frequencies of MNBN cells that were generally similar to and not significantly different (at the p≤0.05 level), compared to those observed in the concurrent vehicle controls, at any concentration analysed under each treatment condition. The MNBN cell frequencies fell within the normal ranges at all concentrations analysed with the exception of one culture at the highest concentration analysed following the 24+24 hour treatment in the absence of S9 (1% at 200 µg/mL, which gave 55% mean cytotoxicity). However, there were no statistically significant increases in MNBN cell frequency at any concentration analysed following the 24+24 hour treatment in the absence of S9 and no statistically significant linear trend, therefore this isolated observation was considered not biologically relevant. A statistically significant linear trend (p≤0.05) was observed following the 3+21 hour treatment in the presence of S9 but as there were no statistically significant increases in MNBN cell frequency at any concentration analysed, this observation was also considered not biologically relevant.

 

It was concluded that the test material (Kalama® Benzaldehyde FCC grade) did not induce biologically relevant increases in the frequency of micronuclei when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S9) and for 24+24 hours in the absence of S9 under the experimental conditions described.