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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 March 1994 to 13 April 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions. The study was performed on a structurally similar substance the results of which are read-across and considered to be representative of the effects of the registered substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 87/302, L 133, p. 61-63
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA 40 CFR; Ch. I; Part 798; Detection of gene mutation in somatic cells in culture; pp. 717-720 (7-1-86 Edition)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: CHS, cyclohexyl salicylate
- Physical state: Clear colourless liquid
- Storage condition of test material: at room temperature
- Molecular weight (if other than submission substance): 220.3

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HRPT locus)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) supplemented with 10 % foetal calf serum.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes (HAT medium)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment I
Without S9 mix: 0, 0.75, 2.5, 5.0, 7.5 and 8.5 µg/mL
With S9 mix: 0, 3.0, 6.0, 10, 30 and 60 µg/mL

Experiment II
Without S9 mix: 0, 1.0, 2.5, 5.0, 7.5 and 10 µg/mL
With S9 mix: 0, 6.0, 10, 30 and 60 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:ethanol
- Justification for choice of solvent/vehicle: The vehicle was selected by the solubility of the test material in the vehicle and the non-toxic properties of the vehicle to the cell culture.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium. Plastic culture flasks were seeded with approximately 1.5 x 10⁶ (single culture) and 5 x 10² cells (in duplicate) into MEM with 10 % FCS. After 24 hours, the culture medium was replaced with serum free medium containing the appropriate test material concentration either with or without 50 µL/mL of S9 mix. After 4 hours this was replaced with complete medium after washing with saline G twice. All cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO₂.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 1 week. Cultures were treated on day 2 and were transfered to selective medium on day 9
- Selection time (if incubation with a selection agent): 1 week and 10 days for experiment I and II, respectively. Flasks seeded on day 9 of the experiment were fixed and stained on day 16 (experiment I) and day 18 (experiment II)
- Fixation time (start of exposure up to fixation or harvest of cells): Day 16 and day 18 for experiments I and II, respectively.

SELECTION AGENT: 11 µg/mL thioguanine
STAIN: 10 % methylene blue in 0.01 % KOH solution.

NUMBER OF REPLICATIONS:
Mutagenicity was evaluated in 5 replicate flasks per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency.
Evaluation criteria:
The test was considered positive if either a concentration related increase of the mutant frequency or a reproducible positive response for one of the concentrations was observed.
The test was considered non-mutagenic if neither a concentration related increase in the mutant frequency was observed, or if a response observed at one test point was found not to be reproducible.
A response was considered significant if the test material produces a reproducible response at one of the concentrations with a mutant frequency three times higher than the spontaneous mutant frequency in the experiment.
The test material is classified as mutagenic if there is a reproducible concentration-related response of the mutant frequency. A concentration-related response may be considered if a threefold increase was not observed in the mutant frequency (any such result would be compared with the negative control data).

The stained colonies with more than 50 cells were counted. Were colony size was uncertain, the colony was checked using a microscope.
Statistics:
No adequate statistical evaluation was available.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Neither a reproducible relevant increase in mutations was observed not a dose dependent increase in mutations were observed
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Without metabolic activation, the test material was cytotoxic in both experiments at the two highest concentrations tested.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test material was evaluated in a pre-test on toxicity. Toxicity of the test material was evidenced by a reduction in cloning efficiency when tested up to 60.00 µg/mL (the limit of solubility). Based on the results of the pre-test, the concentrations for the definitive tests were selected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without metabolic activation, elevated toxicity was observed in the cloning efficiency of the cells with concentrations of 7.5 and 8.5 µg/mL in experiment I. The cell density at the first subcultivation was also reduced for these two concentrations (54.2 and 31.4 % of the solvent control value).

In experiment II, the cloning efficiency of the cells was considerably reduced in the two upper concentrations without S9 mix. The cell density at first subcultivation was reduced to 61.2 % of the solvent control. The 10.0 µg/mL treatment group could not be evaluated due to the severe reduction in the cell density (4.0 % of the solvent control) at the first subcultivation.

In the presence of metabolic activation, no toxic effects were observed up to 60.0 µg/mL (the limit of solubility) in the cloning efficiency of either experiment. No reduction in cell culture at first subcultivation was obtained in experiment I, and only a slight reduction was observed at 60.0 µg/mL in experiment II.

Any other information on results incl. tables

Table 1: Toxicity results from mutagenicity experiment 1

Treatment group

Number of cells per flask

Factor calculated*

Cells seeded

Cells survived**

Seeded (I/II)

Found I

Found II

Mean

Without S9 mix

Negative control

501

360

334

347.0

0.69

438000

302220

Solvent control with ethanol

506

310

341

325.5

0.64

441000

282240

Positive control with 0.60 mg EMS

630

356

396

376.0

0.60

489000

293400

0.75 µg

505

344

356

350.0

0.69

421500

290835

2.50 µg

Culture not continued

5.00 µg

503

334

316

325.0

0.65

423000

274950

7.50 µg

506

319

330

324.5

0.64

427500

273600

8.50 µg

507

312

301

306.5

0.60

436500

261900

With S9 mix

Negative control

506

358

330

344.0

0.68

493000

335240

Solvent control with ethanol

504

322

306

314.0

0.62

354000

219480

Solvent control with DMSO

500

310

298

304.0

0.61

484500

295545

Positive control with 3.85 µg DMBA

506

295

337

316.0

0.62

411000

254820

3.00 µg

Culture not continued

6.00 µg

501

314

338

326..0

0.65

435000

282750

10.00 µg

500

329

321

325.0

0.65

470500

305825

30.00 µg

508

362

344

353.0

0.69

414000

285660

60.00 µg

503

302

328

315.0

0.63

427000

269010

*Factor calculated (mean/cells seeded I/II).

** Cells survived after plating in TG containing medium

Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Table 2:Mutagenicity results from mutagenicity experiment 1

Treatment group

Number of mutant colonies per flask* found after plating in TG medium

Mutant colonies per 106cells

I

II

III

IV

V

Mean

Standard deviation

Without S9 mix

Negative control

3

3

2

2

0

2.0

1.2

6.6

Solvent control with ethanol

2

1

3

2

1

1.8

0.8

6.4

Positive control with 0.60 mg EMS

136

143

153

129

168

145.8

15.3

496.9

0.75 µg

1

1

0

1

1

0.8

0.4

2.8

2.50 µg

Culture not continued

5.00 µg

7

5

3

7

5

5.4

1.7

19.6

7.50 µg

2

1

1

0

1

1.0

0.7

3.7

8.50 µg

1

1

2

2

1

1.4

0.5

5.3

With S9 mix

Negative control

4

2

5

2

4

3.4

1.3

10.1

Solvent control with ethanol

7

9

5

0

3

4.8

3.5

21.9

Solvent control with DMSO

4

3

2

1

2

2.4

1.1

8.1

Positive control with 3.85 µg DMBA

91

85

98

112

107

98.6

11.1

386.9

3.00 µg

Culture not continued

6.00 µg

6

5

2

2

7

4.4

2.3

15.6

10.00 µg

3

2

2

2

3

2.4

0.5

7.8

30.00 µg

2

1

3

3

2

2.2

0.8

7.7

60.00 µg

5

4

1

3

9

4.4

3.0

16.4

Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Table 3: Toxicity results from mutagenicity experiment 2

Treatment group

Number of cells per flask

Factor calculated*

Cells seeded

Cells survived**

Seeded (I/II)

Found I

Found II

Mean

Without S9 mix

Negative control

503

301

318

309.5

0.62

372000

230640

Solvent control with ethanol

507

363

318

340.5

0.67

375000

251250

Positive control with 0.60 mg EMS

505

295

302

298.5

0.59

393000

231870

1.00 µg

504

292

324

308.0

0.61

369000

225090

2.50 µg

509

291

273

282.0

0.55

348000

191400

5.00 µg

505

275

277

276.0

0.55

378000

207900

7.50 µg

510

259

255

257.0

0.50

411000

205500

10.00 µg

Not possible to evaluate, toxic to cultures

With S9 mix

Negative control

510

390

367

378.5

0.74

375000

277500

Solvent control with ethanol

507

302

340

321.0

0.63

363000

228690

Solvent control with DMSO

505

341

287

314.0

0.62

366000

226920

Positive control with 3.85 µg DMBA

510

301

310

305.5

0.60

360000

216000

6.00 µg

504

340

362

351.0

0.70

378000

264600

10.00 µg

515

388

360

374.0

0.73

366000

267180

30.00 µg

511

323

337

330.0

0.65

369000

239850

60.00 µg

511

265

270

267.5

0.52

390000

202800

*Factor calculated (mean/cells seeded I/II).

** Cells survived after plating in TG containing medium

Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Table 4: Mutagenicity results from mutagenicity experiment 2

Treatment group

Number of mutant colonies per flask* found after plating in TG medium

Mutant colonies per 106cells

I

II

III

IV

V

Mean

Standard deviation

Without S9 mix

Negative control

0

1

1

1

1

0.8

0.4

3.5

Solvent control with ethanol

3

4

1

4

3

3.0

1.2

11.9

Positive control with 0.60 mg EMS

93

81

51

61

69

71.0

16.5

306.2

1.00 µg

1

0

0

0

2

0.6

0.9

2.7

2.50 µg

1

2

1

1

0

1.0

0.7

5.2

5.00 µg

1

2

4

0

0

1.4

1.7

6.7

7.50 µg

0

5

5

1

1

2.4

2.4

11.7

10.00 µg

Not possible to evaluate, toxic to cultures

With S9 mix

Negative control

1

5

3

3

5

3.4

1.7

12.3

Solvent control with ethanol

7

3

1

1

3

3.0

2.4

13.1

Solvent control with DMSO

2

1

0

4

1

1.6

1.5

7.1

Positive control with 3.85 µg DMBA

90

103

96

86

95

94.0

6.4

435.2

6.00 µg

1

3

1

3

3

2.2

1.1

8.3

10.00 µg

1

0

0

1

1

0.6

0.5

2.2

30.00 µg

5

4

3

1

3

3.2

1.5

13.3

60.00 µg

3

1

2

3

0

1.8

1.3

8.9

Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Applicant's summary and conclusion

Conclusions:
negative with and without metabolic activation

Under the conditions of the test, the test material did not induce gene mutations at the HRPT locus in V79 cells.
Executive summary:

The ability of the test material to induce mutations at the HPRT locus in Chinese Hamster V79 cells was investigated. In two independent experiments, both performed with and without metabolic activation. The test material was dissolved in ethanol and tested in the following concentrations:

Experiment I:

Without S9 mix: 0.75, 2.5, 5.0 7.5 and 8.5 µg/mL

With S9 mix: 3.0, 6.0 10, 30 and 60 µg/mL

Experiment II:

Without S9 mix: 1.0, 2.5, 5.0, 7.5 and 10.0 µg/mL

With S9 mix: 6.0, 10, 30 and 60 µg/mL

Doses were selected based on a pre-test for toxicity. Without S9 mix, the highest concentration demonstrated strong toxicity. With S9 mix, no toxicity was observed up to the limit of solubility (60 µg/mL).

No relevant increase in mutant colony numbers were obtained in either experiment with or without metabolic activation. The positive negative, and vehicle controls were all demonstrated to be valid under the conditions of the test.

Under the conditions of the test, the test material did not induce gene mutations at the HRPT locus in V79 cells.