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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 from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
February 01st, 1999 - April 21st, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance 131459-39-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
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 the purity of the test substance
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No details on the purity of the test substance
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: straw colored liquid
- Storage condition of test material: RT in the dark

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced S9-Mix from male Spraque-Dawley rats
Test concentrations with justification for top dose:
1. Experiment:
4(20) h without S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml
4(20) h with S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml

2. Experiment
24 h without S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml
4(20) h with S9: 0; 39.06; 78.13; 156.25; 312,5; 625; 1250; 2500; 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in absence of S9-Mix

Migrated to IUCLID6: 750 µg/ml
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in presence of S9-Mix

Migrated to IUCLID6: 25 µg/ml
Details on test system and experimental conditions:
Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels. For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of approx. 14 h was determined by BrdU incorporation.
METHOD OF APPLICATION: in medium (Eagle´s minimal essential medium with HEPES buffer supplemented with L-glutamine, Pen/Strep, amphotericin B and 15% FCS

DURATION
- Exposure duration: Experiment I: 4 h with or without S9 mix and 20 h expression time.
Experiment II: Either 4 h with S9 mix and 20 h expression time or 24 h without S9 mix.
- Expression time (cells in growth medium): 20 h after 4 h exposure to the test substance


SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 min

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER: Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations at up to three dose levels. For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of approx. 14 h was determined by BrdU incorporation.
Evaluation criteria:
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
Statistics:
The frequency of cells with aberrations and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher´s Exact test.

Results and discussion

Test resultsopen allclose all
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
at and above 1250 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No detectable effects
- Effects of osmolality: No detectable effects
- Precipitation: Medium was "cloudy" from 156.25 µg/ml and a precipitate was seen from 1250 µg/ml without effects on the toxicity responsefects:

COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: strain/cell type: lymphocytes
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1: 4 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

5

0

1

0

1

0

7

2

7

2

1250

200

5

0

0

3

0

0

8

3

8

3

2500

200

2

2

0

0

0

0

4

2

4

2

5000

200

5

4

0

0

1

0

10

5

9

5

EMS 750

200

24

9

5

5

0

0

43

19

35

17

Experiment 1: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

4

1

0

0

0

0

5

1

4

1

1250

200

6

4

0

0

0

0

10

4

9

4

2500

200

4

2

0

0

0

0

6

2

5

2

5000

200

2

4

0

1

0

0

7

5

7

5

CP 25

200

13

12

3

2

0

0

30

17

24

15

Experiment 2: 20 h treatment without metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

2

2

1

0

0

0

5

3

5

3

1250

200

7

0

0

1

0

0

8

1

8

1

2500

200

0

1

0

0

0

0

1

1

1

1

5000

200

6

0

0

0

0

0

6

0

6

0

EMS 500

200

53

41

13

2

0

0

109

56

74

48

Experiment 2: 4 h treatment with metabolic activation

Treatment group (µg/ml)

Number of cells scored

Total gaps

Chromatid

Chromosome

others

Total abberations

Abberant cells

Breaks

Exchanges

Breaks

Exchanges

x

(+Gaps)

(+Gaps)

(+Gaps)

(+Gaps)

Vehicle control

200

0

0

0

2

0

0

2

2

2

2

1250

200

4

2

2

3

0

0

9

5

7

3

2500

200

5

2

0

1

0

0

8

3

8

3

5000

200

3

2

0

2

0

0

7

4

6

3

CP 25

200

10

10

2

0

0

0

22

12

15

11

The test substance was non-clastogenic to human lymphocytes in vitro.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative non-clastogenic to human lymphocytes in vitro