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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Sep - 03 Nov 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: Basic mutagenicity tests: UKEMS recommended procedures
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Vinasses, residue of fermentation
EC Number:
932-215-9
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Vinasses, residue of fermentation
Details on test material:
- Name of test material (as cited in study report): Lucullus 235/02
- Physical state / appearance: brown pasty mass
- Analytical purity: 100%
- Lot/batch No.: Lot 235/04
- Storage condition of test material: 4°C

Method

Species / strain
Species / strain / cell type:
other: human lymphocytes (of two healthy donors)
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 20% fetal calf serum, 1% L-glutamine (200 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and fungison (0.25 µg/mL) [+3.6% phytohaemagglutinin]
Additional strain / cell type characteristics:
other: karyotype is stable (46 chromosomes), cell cycle time of 12-14 hours, low spontaneous incidence of abberant cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix , S9 fraction comes from liver homogenates from rats induced with Aroclor 1254 (500 mg/kg)
Test concentrations with justification for top dose:
first test: with and without S9 mix: 0, 156.25, 312.5, 625, 1250, 2500, 5000 µg/mL
repeat test: with and without S9 mix: 0, 1250, 2500, 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Concentration: 280 mg/mL
Controls
Untreated negative controls:
yes
Remarks:
untreated culture
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycine C (MMC) without S9 mix and cyclophosphamide (CPA) with S9 mix
Remarks:
MMC: 0.2 µg/mL; CPA: 50 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9 mix: 24 or 48 hours; with S9 mix: 2 hours
- Expression time (cells in growth medium): about 1.5 normal cell cycle times after the beginning of the treatment (repeat test: additional sample approximately 24 hours later)
- Fixation time (start of exposure up to fixation or harvest of cells): with and without S9 mix: 24 or 48 hours (with S9 mix: 2 h + 22 h; 2 h + 46 h)

SPINDLE INHIBITOR (cytogenetic assays): colcemid solution (0.2 µg/mL), 2h prior to harvesting
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: The number of cells in mitosis was evaluated on a total of 1000 cells. 200 metaphases/concentration were analysed whenever possible (for the positive controls, only 100 metaphases/concentration were scored)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
For determining a positive response:
- a reproducible and statistically significant increase in the aberrant cell frequency for at least one of the tested concentrations

A test substance was considered as non-clastogenic in this test system if there is no significant increase in aberrant cell frequency at any dose above concurrent control frequencies and in both of the two tests and the two harvest times.

Both biological and statistical significance was considered together in the evaluation.
Statistics:
For each test and for each harvest time, the incidence of aberrant cells (excluding gaps) in treated cultures was compared to that of the solvent cultures. The comparison was performed using the Chi-square test, in which p= 0.05 was used as the lowest level of significance.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no toxicity; the mitotic index was reduced by 0 - 40% to that of controls
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitate was observed (up to 5000 µg/mL)

COMPARISON WITH HISTORICAL CONTROL DATA: For both tests, the incidence of aberrant cells in the negative and solvent controls was within the range of historical data (i.e., 0.8 +/- 0.5%, gaps excluded).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The incidence of aberrant cells in the cultures treated with the test substance LUCULLUS 235/02- Batch No. 235/04 was similar to that of the negative controls in the first and repeat tests including an additional harvest 24 hours later.

The test substance did not show clastogenic activity in this chromosomal aberration test performed in cultured human lymphocytes.

Table 1: Lucullus 235/02 Batch No. 235/04: First clastogenicity test with and without S9 mix (fixation interval: 24 hours)

First test

fixation interval of 24 hours

S9 mix

aberrant cells (% mean)

incl. gaps

aberrant cells (% mean)

excl. gaps

Mitotic index relative (%) of the control

negative control

-

0

0

100

positive control

-

39

38

55

156.25 µg/mL

-

-

-

150

312.5 µg/mL

-

-

-

136

625 µg/mL

-

-

-

110

1250 µg/mL

-

0

0

148

2500 µg/mL

-

0.7

0

155

5000 µg/mL

-

0.5

0.5

119

negative control

+

0.5

0.5

100

positive control

+

65.0

65.0

14

156.25 µg/mL

+

-

-

74

312.5 µg/mL

+

-

-

91

625 µg/mL

+

-

-

91

1250 µg/mL

+

0

0

80

2500 µg/mL

+

1.5

1.0

91

5000 µg/mL

+

0.5

0.5

82

200 metaphases were scored per test concentration (except 2500 µg/mL without S9 mix only150) and 100 metaphases were scored for the positive control.

Table 2: Lucullus 235/02 Batch No. 235/04: Repeat clastogenicity test with and without metabolic activation (fixation interval: 24 hours)

Repeat test

fixation interval of 24 hours

S9 mix

aberrant cells (% mean)

incl. gaps

aberrant cells (% mean)

excl. gaps

Mitotic index relative (%) of the control

negative control

-

1.0

0.5

100

positive control

-

30

30

32

1250 µg/mL

-

0

0

96

2500 µg/mL

-

0

0

52

5000 µg/mL

-

0.5

0.5

60

negative control

+

1

0.5

100

positive control

+

33

33

52

1250 µg/mL

+

0.5

0.5

73

2500 µg/mL

+

0

0

77

5000 µg/mL

+

0

0

83

200 metaphases were scored per test concentration and 100 metaphases were scored for the positive control (except control without S9 mix only 90 metaphases were scored for)

Table 3: Lucullus 235/02 Batch No. 235/04: Repeat clastogenicity test with and without metabolic activation (fixation time of 48 hours)

Repeat test

fixation interval of 48 hours

S9 mix

aberrant cells (% mean)

incl. gaps

aberrant cells (% mean)

excl. gaps

Mitotic index relative (%) of the control

negative control

-

0

0

100

1250 µg/mL

-

0

0

88

2500 µg/mL

-

0.5

0.5

112

5000 µg/mL

-

0

0

60

negative control

+

0.5

0

100

1250 µg/mL

+

0

0

74

2500 µg/mL

+

0.5

0.5

69

5000 µg/mL

+

0

0

65

200 metaphases were scored per test concentration.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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