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Toxicological information

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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
23 Feb - 26 Jul 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no details on analytical purity of the test substance given).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no details on analytical purity of the test substance given
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no details on analytical purity of the test substance given
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
yes
Remarks:
no details on analytical purity of the test substance given
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MRD-99-429
- Physical state: pale yellow liquid
- Analytical purity: no data
- Expiration date of the lot/batch: 09/2004 (container 1) and 03/2005 (container 2)
- Storage condition of test material: at room temperature

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: in the absence of S9 mix: McCoy's 5A Medium containing 10% foetal bovine serum and 2 mM L-glutamine; in the presence of S9 mix: serum-free McCoy's 5A Medium containing 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats treated with Aroclor
Test concentrations with justification for top dose:
Range-finder toxicity test: 0, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL (3 h treatment), with and without S9
Initial and repeat experiment (main assay): 25, 75, 250, 750 and 2500 µg/mL (3 h treatment), with and without S9
Chromosome aberration analysis: 75, 750, and 2500 µg/mL (without S9); 25, 250 and 2500 µg/mL (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: a vehicle solubility test was previously performed to assess the solubility of the test substance in DMSO, water and acetone. The results of this test showed that the test substance was soluble in acetone at the concentrations required for this study. The non-cytotoxic dose volume for acetone is 50 µL/flask, therefore based on solubility, 2500 µg/mL was the highest dose that could be achieved in this assay.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
- S): 9,10-dimethyl- 1,2-benzanthracene (DMBA, 10 µg/mL in acetone); + S9: 1-methyl-3-nitro-1-nitrosoguanidine (MNNG, 0.6 µg/mL in acetone
Positive control substance:
9,10-dimethylbenzanthracene
other: 1-methyl-3-nitro-1-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation or harvest of cells):
A) Range-finder toxicity test: 19 h
B) Initial experiment (main assay): 19 h
C) Repeat experiment (main assay): 19 and 43 h

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL Colcemid®
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell confluency (range-finder toxicity test and main assay); other: cell count (range-finder toxicity test)
Evaluation criteria:
For a test substance to be considered as positive, one of the following conditions must be met:
- a statistically significant dose-related increase in the mean percentage of aberrant cells and, in at least one of the treatment groups, the mean percentage of aberrant cells exceeds 5%.
OR
- a reproducible and statistically significant response for at least one of the treatment groups is observed. In addition, the mean percentage of aberrant cells exceeds 5%.
A positive result indicates that the test substance induces chromosomal aberrations in cultured mammalian somatic cells. If neither of the above conditions are met, the test substance is considered as non-mutagenic in this system.
Statistics:
The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were used for statistical analysis. The number of aberrant individual chromosomes per cell was not statistically analysed.

The results of the positive control group were compared to the appropriate vehicle control group by the Fisher Exact Test to assure that the assay was performed in an appropriate manner.

To test for homogeneity of the replicates, each pair of replicates was compared by Fisher Exact Test. Two times the sum of the log of the individual two-sided significance levels was compared to chi-square distribution with 2k degrees of freedom (k is the number of replicate pairs). If the test failed, further investigation would be pursued and the remaining analyses would not be performed.

To test for differences among the control and the treated groups, a 2x2-Fisher Exact Test was performed. If differences were shown to exist at the 0.05 level or less, individual 2x2-Fisher Tests were performed to determine which of the treated groups differed from the control group. A permutation test (Hoeffding, 1952) was performed to test for dose-related trends. Statistical analysis included the calculation of means for percent confluency, percent mitotic cells, percent aberrant cells, percent frequency of aberrations, and cell counts. Significance was reported when p < 0.05.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: in the range-finder toxicity test, visual solubility observations were made 3 h after treatment. Cloudiness was evident at all concentrations with S9. Complete solubility was observed at concentrations ranging from 10 to 39 µg/mL without S9. Cloudiness was evident at concentrations of 78 µg/mL through 625 µg/mL without S9. Oil droplets were noted without metabolic activation at concentrations ≥ 1250 µg/mL. A notable decrease in confluency (≥ 50% reduction compared with vehicle controls) were observed at concentrations of 625 µg/mL and 1250 µg/mL with S9 and 2500 µg/mL without S9. Decreases in cell survival and mitotic index (MI) of ≥ 50% from the vehicle control were not observed in either the activated or non-activated series. Cell morphology was normal at all concentrations tested, however debris of undetermined origin was noted in activated flasks at concentrations ≥ 156 µg/mL and non-activated flasks ≥ 313 µg/mL. Based on these results, 25, 75, 250, 750 and 2500 µg/mL were selected as concentrations for the main chromosomal aberration assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in both experiments of the main study, there was no notable decrease in the percent confluency (≥ 50% reduction compared to the vehicle control) at any concentration with or without S9. Cell morphology was normal at all concentrations tested. Similarly, no notable decrease (≥ 50%) in MI values was noted for the test substance concentrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

CHROMOSOME ANALYSIS

A) INITIAL ASSAY

There were no statistically significant differences or dose-related trends in the percentage of cells with chromosomal aberrations between the treated and the control groups either with or without metabolic activation. The percentage of aberrant cells in the vehicle control groups, with and without metabolic activation, was 2.5 and 1.0%, respectively. Both values fell within the 0 to 5% range of acceptance for the vehicle control. The percentage of aberrant cells in the treatment groups ranged from 2.0 to 3.0% for the metabolically activated series and from 0.5 to 3.0% in the non-activated series.

Table 1. Test results of the initial experiment

Test item

Concentration in µg/mL

Mitotix index in % of vehicle control

Aberrant cells in % per 200 cells scored

Frequency of abberations in % per 200 cells scored

with gaps

without gaps

Exposure period 3 h, fixation time 19 h, without S9 mix

Acetone

a

100

1.0

1.5

1

MNNG

0.6

26

12.0**

5.0

12.5

Test substance

75

77

0.5

2.5

0.5

750

80

3.0

1.0

3

2500

54

2.0

0.0

2

Exposure period 3h, fixation time 19 h, with S9 mix

Acetone

a

100

2.5

2.5

3.0

DMBA

10

59

19.0**

5.0

26.0

Test substance

25

112

3.0

3.0

3.0

250

100

2.5

1.5

2.5

2500

104

2.0

3.0

2.0

MNNG = 1-methyl-3-nitro-1-nitrosoguanidine; DMBA = 9,10-dimethyl-1,2-benzanthracene

a = concentration: 50 µL/flask; b = positive controls were not required for the 43 h harvest

*p < 0.05; **p < 0.01

B) REPEAT ASSAY

There was a statistically significant difference (p < 0.05) in the percentage of cells with chromosomal aberrations between the vehicle and the highest concentration (2500 µg/mL) in the 19 h harvest with metabolic activation. The permutation test for the 19 h harvest with metabolic activation also appears to be significant (p<0.01) indicating a dose-related trend in the percentage of cells with chromosomal aberrations. This increase in the percentage of aberrant cells does not appear to be biologically significant due to the fact that the percentage of aberrant cells fell within the acceptable range for the vehicle control (0-5%).

The percentage of aberrant cells in the vehicle control groups at both harvest intervals (with and without metabolic activation) was 2.5% or less. These values fell within the 0 to 5% range of acceptance for the vehicle control. The percentage of aberrant cells observed in the 19 h treated groups ranged from 1 to 3.0%. The percentage of aberrant cells observed in the 43 h treated groups ranged from 0.5% to 2.5%.

Table 2. Test results of the repeat experiment

Test item

Concentration in µg/mL

Mitotix index in % of vehicle control

Aberrant cells in % per 200 cells scored

Frequency of abberations in % per 200 cells scored

with gaps

without gaps

Exposure period 3 h, fixation time 19 h, without S9 mix

Acetone

a

100

1.0

1.0

1.0

MNNG

0.6

42

8.0**

2.0

13.5

Test substance

75

89

2.0

2.5

2.5

750

65

1.5

1.0

1.5

2500

82

1.5

1.0

2.0

Exposure period 3 h, fixation time 43 h, without S9 mix

Acetone

a

100

2.5

2.5

2.5

MNNG

0.6

b

b

b

b

Test substance

75

105

0.5

1.0

0.5

750

95

0.5

1.5

0.5

2500

68

1.5

2.0

1.5

Exposure period 3 h, fixation time 19 h, with S9 mix

Acetone

a

100

0.0

1.5

0.0

DMBA

10

66

10.5**

7.0

11.5

Test substance

25

107

1.0

1.0

1.0

250

105

1.0

1.0

1.0

2500

60

3.0* #

1.0

3.5

Exposure period 3 h, fixation time 43 h, with S9 mix

Acetone

a

100

1.0

1.0

1.0

DMBA

0.6

b

b

b

b

Test substance

25

89

2.5

2.5

2.5

250

70

1.5

1.0

1.5

2500

68

2.0

0.0

2.0

MNNG = 1-methyl-3-nitro-1-nitrosoguanidine; DMBA = 9,10-dimethyl-1,2-benzanthracene

a = concentration: 50 µL/flask; b = positive controls were not required for the 43 h harvest

*p < 0.05; **p < 0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative