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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
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Metabolic activation was achieved by addition of S-9 mix, which consisted of the S-9 fraction from the liver of male Wistar Hanlbm rats treated with Phenobarbital i.p. and ß-Naphthoflavone p.o., mixed with a series of cofactors.
Test concentrations with justification for top dose:
2.5, 5.0, 10.0, 20.0, 30.0, and 40.0 µg/mL (with and without S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water, nutrient medium
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate and Cyclophosphamide
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 18 hours
- Selection time (if incubation with a selection agent): 14 hours

SELECTION AGENT (mutation assays): Chromosomes

NUMBER OF CELLS EVALUATED: 1E+06

DETERMINATION OF CYTOTOXICITY:
Toxicity of the test substance is indicated by a reduction of mitotic indeces.

S9 mix Preparation:
Metabolic activation was achieved by addition of S-9 mix, which consisted of the S-9 fraction from the liver of male Wistar Hanlbm rats treated with Phenobarbital i.p. and ß-Naphthoflavone p.o., mixed with a series of cofactors (MgCl2, KCl, Glucose-6-phosphate, NADP in sodium-ortho-phosphate buffer).

Dose Selection:
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. 3000 µg/mL of the aqueous test substance solution (corresponds to approx . 10 mM of the active ingredient) were applied as top concentration for treatment of the cultures in the pre-test. Test substance concentrations between 23.4 and 3000 µg/mL (with and without S9-mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, in the absence of S9 mix only precipitation of the test substance after 4 hrs treatment was observed at 1500 µg/mL and above.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 23.4 µg/mL and above in the absence and the presence of S9 mix. In addition, 24 hrs continuous treatment with 23.4 µg/ml and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 40 µg/mL (with and without S9 mix) were chosen as top concentrations in the main experiment. Since the test substance was considered to be clastogenic after 4 hrs treatment a second experiment was not performed.

Negative Controls:
Concurrent negative (culture medium) and solvent controls (deionised water) were performed.

Positive Controls:
- Without metabolic activation: Ethylmethane sulfonate (EMS), purity: >98%, dissolved in: nutrient medium, final conc.: 400 µg/mL = 3.2 mM
- With metabolic activation: Cyclophosphamide (CPA), purity: approx. 98%, dissolved in: nutrient medium, final conc.: 0.7 µg/mL = 2.5 µM
Evaluation criteria:
A test substance is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the historical control data of the performing laboratory (0.0 - 4.0 % aberrant cells exclusive gaps).

A test substance is classified as clastogenic if :
- the number of induced structural chromosome aberrations are not in the range of the historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

However, both biological and statistical significance should be considered together . If the criteria mentioned above for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test substance can be classified as aneugenic if:
- the number of induced numerical aberrations are not in the range of the historical control data (0.0 % - 8.5 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher"s exact test (Ricardson C. et al., 1989): p < 0.05.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: 30 µg/mL without S9-mix; 40 µg/mL with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Analysis of Aberrations (200 cells analyzed); preparation interval 18 hrs without S9 mix: exposure period 4 hrs.

Test Substance

Dose

(µg/mL)

% Aberrant cells

(excl. gaps)

Chromatid type

Chromosome type

Break

Exchange

Break

Exchange

Negative Control

-

2.0

2

0

1

1

Solvent Control

Deion. water 10 % (v/v)

0.5

0

1

0

0

Positive Control

EMS 400

47.5

52

47

9

0

Protectol HT

10

7.5*

6

8

2

0

20

25.5*

19

46

5

0

30

43.0*

23

83

15

0

Analysis of Aberrations (200 cells analyzed); preparation interval 18 hrs with S9 mix: exposure period 4 hrs.

Test Substance

Dose

(µg/mL)

% Aberrant cells

(excl. gaps)

Chromatid type

Chromosome type

Break

Exchange

Break

Exchange

Negative Control

-

2.5

3

0

0

0

Solvent Control

Deion. water 10 % (v/v)

2.0

3

0

1

1

Positive Control

CPA 0.7

16.5

15

18

2

0

Protectol HT

5

2.0

2

0

0

0

10

3.0

2

2

2

0

20

15.0*

13

18

4

0

* Aberration frequency statiscally significant higher than corresponding control values.

Applicant's summary and conclusion