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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (shorter exposure period. Lack of data on test substance, no positive controls for 40 h time point)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
(in both experiments, cultures without metabolic activation were exposed to the test substance for about 16 h, no positive control for the 40 h time point)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

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: McCoy's 5A Medium containing 10% (v/v) fetal bovine serum and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
other: WBL clone
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
40, 80 and 160 µg/mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on results of a solubility test, acetone was selected as the vehicle. The test substance was not soluble in water or dimethyl sulfoxide at any of the concentrations (10, 25, 50% (v/v)) tested. The test substance was soluble as a 50% mixture in acetone.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL (v/v) in acetone; + S9: 7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL (v/v) in acetone
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
+S9: ca. 3 h (± 0.5 h)
- S9: ca. 16 h (± 0.5 h)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 16 h (± 0.5 h); second experiment - ca. 16 and 40 h (± 0.5 h)

SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL Colcemid® (10 mg/mL (v/v) in cell culture medium)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 replications (16 h) and 1 replication (40 h), respectively

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
Evaluation criteria:
A test substance was considered positive in the chromosome aberration test if:
1. A statistically significant dose-related increase in the percentage of aberrant cells and in at least one of the treatment groups, the percentage of aberrant cells exceeds 5%. OR
2. 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 under the test conditions the test substance induces chromosomal aberrations in cultured mammalian somatic cells.
If neither of the above conditions exist, the test substance is considered nonmutagenic or negative for inducing chromosomal aberrations in this system.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is not soluble in water, therefore it was dissolved in acetone.
- Precipitation: Final concentrations of the test substance in medium of 10, 20, 39, 78, 156, 313, 625, 1250 and 2500 µg/mL were tested by visual and microscopic methods for precipitation immediately, 30 minutes and 3 h after dosing. Traces of the test substance were observed microscopically at all test concentrations equal to or greater than 78 µg/mL. Therefore, the upper limit of the culture medium solubility of the test substance was considered to be between 39 and 78 µg/mL. Based on these results, the study director selected the following concentrations for the toxicity pretest: 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL.
In the main experiments, slight precipitation was observed in the second experiment after 16 h at 160 µg/mL without metabolic activation. Precipitation was not noted at any other harvest of a 160 µg/mL culture.

RANGE-FINDING/SCREENING STUDIES: To determine a concentration selection for the aberration assay, a toxicity pretest was conducted with concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL of the test substance with and without metabolic activation. The concentrations tested were based on the results of a culture medium solubility test. The cultures with metabolic activation were treated for 3 h (± 0.5 h). The cultures without metabolic activation were treated until 2-3 h prior to harvest. All cultures were harvested about 16 h from the beginning of treatment. After harvest, the number of cells that survived treatment were counted using a hemacytometer to evaluate cytotoxicity and the mitotic indices (number of mitotic cells per 1000 total cells) were determined to evaluate cell cycle suppression. The selected concentrations for the aberration assay were based on the results of the cell count data and mitotic index data. The highest reduction in cell survival was observed at 160 µg/mL without metabolic acvtivation, where reduction in viability of 37% was noted. Other less notable reductions in cell survival were noted (see table 3), but were not indicative of a concentration-related trend. Based on these results, the concentrations selected for the aberration assay were 40, 80 and 160 µg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1

Test item

Concentration

Mitotic Index

Aberrant cells

Aberration frequency

 

 in µg/mL

in %

in %

in %

Exposure period 16 h, fixation time 16 h, without S9 mix

vehicle

0.5% (v/v)

6.8

0.5

0.5

MNNG

0.6

6.2

22.5**

24.5

Test substance

40

5.6

0.5

0.5

80

6.5

0.5

0.5

160

7.2

1.0

1.0

Exposure period 3 h, fixation time 16 h, with S9 mix

Acetone

0.5% (v/v)

5.5

1.0

1.0

DMBA

10

2.6

33.5**

42.0

Test substance

40

5.3

1.5

1.5

80

6.3

0.5

0.5

160

4.6

2.0

2.0

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

 

Table 2. Test results of experiment 2

Test item

Concentration

Mitotic Index

Aberrant cells

Aberration frequency

 

 in µg/mL

in %

in %

in %

Exposure period 16 h, fixation time 16 h, without S9 mix

vehicle

0.5% (v/v)

7.2

1.0

1.0

MNNG

0.6

5.4

17.5**

17.5

Test substance

40

7.1

1.0

1.0

80

5.4

0.5

0.5

160

7.1

2.5

2.5

Exposure period 3 h, fixation time 16 h, with S9 mix

Acetone

0.5% (v/v)

2.2

0.0

0.0

DMBA

10

4.5

33.0**

43.0

Test substance

40

2.2

1.0

1.0

80

2.4

2.0

2.0

160

2.0

1.5

1.5

Exposure period 16 h, fixation time 40 h, without S9 mix

Acetone

0.5% (v/v)

3.4

2.5

2.0

MNNG #

0.6

---

---

---

Test substance

40

3.0

4.0

4.5

80

2.2

3.0

3.0

160

3.4

2.0

2.0

Exposure period 3 h, fixation time 40 h, with S9 mix

Acetone

0.5% (v/v)

4.8

2.5

2.5

DMBA #

10

---

---

---

Test substance

40

5.4

2.0

2.0

80

4.8

2.0

2.0

160

5.0

0.5

0.5

 

**statistically significantly higher than vehicle control (p<0.001)

MNNG: N-Methyl-N-Nitro-N-Nitrosoguanidine; DMBA: 7,12-Dimethylbenz[a]anthracene (positive controls)

# According to the study report, positive controls were not required for the 40 h harvest.

Table 3. Toxicity pretest results

Treatment Group
in µg/mL

Cell Survival in %*

+ S9

- S9

non-treated

107

102

vehicle

100

100

0.625

122

114

1.25

95

72

2.5

121

88

5

108

68

10

111

108

20

89

102

40

99

93

80

78

93

160

120

63

  * % cell survival as compared to vehicle

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative