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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:
10 August 2012 and 07 November 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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:
other: solid
Details on test material:
- Physical state: Pale Yellow Solid
- Purity: Not applicable - UVCB
- Substance identity: Aluminum, benzoate C16-18-fatty acids complexes
- Batch number: 11074091 + Benzoic acid
- Carbon Content: 65.1%
- Analysis code: A118
- Date recieved: 07 June 2012
- Expiration date: 01 July 2013
- Storage of test material: Room temperature in the dark

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
The details of the donors used in this study are:
Preliminary Toxicity Test: Male, aged 27 years
Experiments 1 and 2: Female, aged 32 years

Cell Culture: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction prepared from the liver of male Sprague-Dawley rats that had received daily doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group
4-hour without S9 0*, 75*, 150*, 300*, 600, 900, 1200 (µg test item /mL)
4-hour with S9 (2%) 0*, 75*, 150*, 225*, 300*, 450, 600 (µg test item /mL)
20-hour without S9 0*, 75*, 150*, 300*, 600, 900, 1200 (µg test item /mL)

* = Dose level selected for analysis of binucleate cells for micronuclei
Vehicle / solvent:
Vehicle: Minimal Essential Medium (MEM)
Justification for choice of vehicle: No data reported
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
Cultures incubated for 48 hours at 37ºC, in 5% CO2 in humidified air before exposure.

DURATION
- Exposure duration: 4 hours in the presence and absence of S9; also 20 hours in the absence of S9.
- Recovery time: 28 hours in presence of Cytochalasin B for all treatments.

FIXATIVE: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375 KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC prior to slide making.

STAIN: Stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: Proportions of mono-, bi- and multinucleate cells were evaluated for a minimum of 500 cells per culture. One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.

EVALUATION PROCEDURE: The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity (%) = 100{(CBPIT – 1)/(CBPIC – 1)}
where:
CBPI = No. mononucleate cells + (2 x No. binucleate cells) + (3 x No. multinucleate cells)/Total number of cells
T = test chemical treatment culture
C = vehicle control culture.

OTHER DETAILS ON TEST SYSTEM:
The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle controls, test article preparations and positive controls were performed in duplicate.

For qualitative slide assessment, the slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

From the range-finder experiment, analysising 500 cells per concentration, and using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.
Evaluation criteria:
A toxicologically (clastogenic and aneugenic potential) significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei is less than 0.05 and there is a dose-related increase in the frequency of cells with aberrations which is reproducible.

The criteria for identifying micronuclei was that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei.

Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei can be attached by a fine nucleoplasmic bridge which is approximately no greater than one quarter of the nuclear diameter.







Statistics:
The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on observed numbers of cells with micronuclei, Hoffman et al (2003).
Reference: Hoffman, WP, Garriott, ML, Lee, C. (2003). In vitro micronucleus test. In: Encyclopaedia of Biopharmaceutical Statistics, 2nd edition. S Chow ed. Marcel Dekker, Inc. New York, NY, pp. 463 – 467

Results and discussion

Test results
Key result
Species / strain:
lymphocytes:
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:
valid
Positive controls validity:
not examined
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant change
- Effects of osmolality: ≤ 50 mOs
- Precipitation in Experiment 1: Precipitate was observed in the blood cultures at the end of the exposure period at and above 600 and 300 µg/mL in the absence and presence of S9 respectively. The precipitate was carried through onto the slides and was seen at and above 300 and 150 µg/mL in the absence and presence of S9 respectively. The maximum dose level selected for analysis of binucleate cells was limited to 300 µg/mL in both exposure groups due to the onset of precipitate affecting the cells.
- Precipitation in Experiment 2: A precipitate of the test item was observed in the blood cultures at the end of the exposure period at and above 75 µg/mL and was observed on the slides at and above 300 µg/mL. The qualitative assessment of the slides determined that there were binucleate cells up to the maximum dose tested but due to the effects of precipitate the maximum dose with binucleate cells suitable for scoring was 300 µg/mL.


RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. The maximum dose was the maximum recommended dose level. The maximum dose level selected for the main experiments was restricted due to the onset of precipitate on the slides limiting the ability to accurately score the binucleate cells. The maximum dose selected for the 4-hour exposure groups of Experiment 1 in the absence and presence of S9 was 1200 and 600 µg/mL respectively and was 1200 µg/mL for the 20-hour exposure group in the absence of S9 in Experiment 2.

COMPARISON WITH HISTORICAL CONTROL DATA: For both experiments, the vehicle control cultures had frequencies of cells with micronuclei within the expected range (see Appendix 1 above and tables below). The positive control items induced statistically significant increases in the frequency of cells with micronuclei (see Appendix 1 above and tables below). The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

Remarks on result:
other: Evaluation of micronuclei in binucleate cells

Any other information on results incl. tables

Table 2. CBPI – Experiment 1

4-Hour exposure without S9

4-Hour exposure without S9 (2%)

Dose level (µg/ml)

Replicate

Nucleate cells/500 cells

CBPI

Mean CBPI

% control CBPI

Dose level (µg/ml)

Replicate

Nucleate cells/500 cells

CBPI

Mean CBPI

% control CBPI

Mono

Bi

Multi

Mono

Bi

Multi

0

A

190

230

80

1.78

1.58

0

0

A

126

254

120

1.99

1.96

100

B

344

118

38

1.39

B

130

275

95

1.93

75

A

167

231

102

1.87

1.66

1.12

75

A

264

180

56

1.58

1.79

82

B

311

158

31

1.44

B

118

266

116

2.00

150

A

168

261

71

1.81

1.73

125

150*

A

327

150

23

1.39

1.68

71

B

219

234

47

1.68

B

132

252

116

1.97

300

A

219

240

41

1.64

1.58

99

225*

A

209

253

38

1.66

1.80

83

B

261

219

20

1.52

B

122

287

91

1.94

600P

A

NSB

NSB

NSB

NSB.

NSB

NSB

300P*

A

194

226

80

1.77

1.79

82

B

NSB

NSB

NSB

NSB.

B

162

276

62

1.80

900P

A

NB

NB

NB

NB

NB

NB

450P*

NSB

NSB

NSB

NSB.

NSB

NSB

NSB

B

NB

NB

NB

NB

NSB

NSB

NSB

NSB.

NSB

1200P

A

NB

NB

NB

NB

NB

NB

600P*

NSB

NSB

NSB

NSB.

NSB

NSB

NSB

B

NB

NB

NB

NB

NSB

NSB

NSB

NSB.

NSB

MMC 0.2

A

244

239

17

1.55

1.54

92

CP 5

A

260

234

6

1.49

1.48

50

B

248

237

15

1.53

B

274

219

7

1.47

MMC    = Mitomycin C

CP        = Cyclophosphamide

NB         = No binucleate cells

NSB      = No binucleate cells suitable for scoring

P           = Precipitate observed at the end of exposure period

           = Precipitate observed on the slides

Table 3: CBPI – Experiment 2

20-Hour exposure without S9

Dose level (µg/ml)

Replicate

Nucleate cells/500 cells

CBPI

Mean CBPI

% Control CBPI

Mono

Bi

Multi

0

A

48

326

126

2.16

2.20

100

B

41

300

159

2.25

75P

A

52

328

120

2.14

2.07

89

B

87

325

88

2.00

150P

A

83

314

103

2.04

2.11

93

B

41

325

134

2.19

300P†

A

129

299

72

1.89

1.87

73

B

158

259

83

1.85

600P†

A

NSB

NSB

NSB

NSB

NSB

NSB

B

NSB

NSB

NSB

NSB

900P†

A

NSB

NSB

NSB

NSB

NSB

NSB

B

NSB

NSB

NSB

NSB

1200P†

A

NSB

NSB

NSB

NSB

NSB

NSB

B

NSB

NSB

NSB

NSB

DC 0.075

A

251

206

43

1.58

1.58

48

B

249

214

37

1.58

DC      = Demecolcine

NSB      = No binucleate cells suitable for scoring

P           = Precipitate observed at the end of exposure period

            = Precipitate observed on the slides

Applicant's summary and conclusion

Conclusions:
Interpretation of results Negative with metabolic activation, negative without metabolic activation
Aluminum, benzoate C16-18-fatty acids complexes did not induce statistically significant increase in the frequency of cells with micronuclei in cultured human peripheral blood lymphocytes when tested up to a limit of solubility in both the absence and presence of a rat liver metabolic activation system (S-9). The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. The test method was designed to be compatible with, OECD Guidelines for Testing of Chemicals (2010) No 487: In Vitro Mammalian Cell Micronucleus Test.

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4 hour exposure in the presence and absence of a standard metabolising system (S9, at a 2% final concentration). Experiment 2, used a 20-hour exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of Experiment 1.

 

Results

All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei, indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments. The maximum dose scored for micronuclei was limited by the onset of precipitate affecting the cell morphology and interfering with the cells on the slides.

Conclusion

The test item, aluminum, benzoate C16-18-fatty acids complexes, was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.