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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
Study period:
From 2008-07-16 to 2008-10-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study performed according to OECD test guideline No. 476 and in compliance with GLP with acceptable restrictions. In the absence of S9, in both experiments, the required level of toxicity was not achieved. Precipitate was observed in Exp. I and IIA but not in IIB. A statement from the study Director is attached to this ESR (see "1193806statement18082011".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
In the absence of S9, in both experiments, the required level of toxicity was not achieved. Precipitate was observed in Exp. I and IIA but not in IIB. Statement from the study Director is attached to this ESR.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
In the absence of S9, in both experiments, the required level of toxicity was not achieved. Precipitate was observed in Exp. I and IIA but not in IIB. Statement from the study Director is attached to this ESR.
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
OECD GLP (Inspected on 2nd September 2006 / Signed on 19th January 2007)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state: solid
- Stability under test conditions: at least 7 days when kept at room temperature.
- Storage condition of test material: In the refrigerator (2 - 8 °C), protected from light
- Molecular formula : C16 H28 O
- Molecular weight : 236.4 g/mol

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: About 5 x 105 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; Seromed, 12247 Berlin, Germany) supplemented with 10 % foetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 Og/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany).
The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from Phenobarbital/B-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
- Experiment I: without S9-mix: from 4.7 to 1200 µg/mL (approx. 5.1 mM) / with S9-mix: from 2.4 to 600 µg/mL (approx. 2.5 mM).
- Experiment IIA: without S9-mix: from 0.3 to 75 µg/mL / with S9-mix: from 18.8 to 600 µg/mL.
- Experiment IIB: with S9-mix: from 4.7 to 600 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
500 and 900 µg/mL (4.0 and 7.2 mM)
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1.4 µg/mL (5.0 mM)
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 h with and without S9-mix
Experiment IIA: 18 h without S9-mix / 4 h with S9-mix
Experiment IIB: 4 h with S9-mix
- Expression time (cells in growth medium): 14 h after wash-off (except experiment IIA without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL culture medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 100 metaphases per culture, except for positive controls in Experiment I and II in the absence of S9-mix, where only 50 metaphases were scored per culture.

DETERMINATION OF CYTOTOXICITY
- Method: Cell number and mitotic index
For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hours in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory’s historical control data range and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory’s historical control data range and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory’s historical control data range.
Statistics:
Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence (Exp. I: solvent control pH 7.4 versus pH 7.3 at 1200 µ/mL; Exp. IIA: solvent control pH 7.3 versus and pH 7.3 at 75 µg/mL).
- Effects of osmolality: No relevant influence (Exp. I: solvent control 354 mOsm versus 370 mOsm at 1200 µg/mL; Exp. IIA: solvent control 355 mOsm versus 380 mOsm at 75 µg/mL).
- Evaporation from medium: the test material is not a VOC
- Water solubility: The test substance was not sufficiently soluble in water for water to be used as the vehicle.
- Precipitation: In Experiment I precipitation of the test item in culture medium was observed after 4 hours treatment with 1200 µg/mL in the absence of S9 mix and 300 µg/mL and above in the presence of S9 mix. In Experiment IIA in the presence of S9 mix precipitation occurred after 4 hours treatment with 300 µg/mL and above.
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA: all vehicle and solvent controls were in the range of historical laboratory control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiments I and IIA in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I in the presence of S9 mix no cytotoxicity indicated by reduced cell numbers and/ or reduced mitotic indices of below 50 % of control was observed up to the highest evaluated concentration, where test item precipitation occurred. In Experiment IIA in the presence of S9 mix, 75.0 µg/mL induced unexpected cytotoxicity. In Experiment IIB no cytotoxicity indicated by reduced cell numbers and/ or reduced mitotic indices of below 50 % of control was observed up to the highest applied concentration.

Any other information on results incl. tables

Table 7.6.1/2: Summary of results

Exp.

Test concentration (µg/mL)

Cell number (% of control)

Mitotic indices (% of control)

Endomitotic cells (%)*

Polyploid cells (%)**

Aberrant cells (%)

Incl. gaps**

Excl. gaps**

With exchanges

Exposure period 4 h without S9-mix

I

Solvent control1

100.0

100.0

0.0

3.4

3.0

2.0

0.0

Positive control2#

n.t.

85.0

0.0

5.6

31.0

30.0S

18.0

4.7

110.8

113.3

0.0

3.4

1.5

1.5

0.0

9.4

110.3

111.3

0.0

3.6

3.0

3.0

0.0

18.8

101.9

97.3

0.0

4.3

2.5

1.5

0.0

Exposure period 18 h without S9-mix

IIA

Solvent control1

100.0

100.0

0.1

2.6

2.5

1.5

0.0

Positive control3#

n.t.

81.0

0.0

2.5

36.0

35.0S

6.0

4.7

91.0

93.2

0.0

1.8

1.0

1.0

0.0

9.4

106.4

113.3

0.1

3.0

1.0

1.0

0.0

18.8

66.2

101.7

0.0

2.5

1.0

1.0

0.5

Exposure period 4 h with S9-mix

I

Solvent control1

100.0

100.0

0.1

4.8

2.5

2.0

1.0

Positive control4#

n.t.

69.6

0.0

2.9

19.5

19.0S

4.5

75.0

97.1

92.7

0.1

3.9

2.5

2.0

0.0

150.0

92.9

96.0

0.0

4.5

2.5

2.0

0.0

300.0

98.9

93.4

0.0

4.9

2.5

1.5

0.0

IIA

Solvent control1

100.0

100.0

0.1

2.9

1.0

1.0

0.0

Positive control4#

n.t.

73.8

0.0

2.6

22.0

21.5S

10.0

37.5

103.7

100.6

0.1

2.2

2.0

1.5

0.5

75.0

31.6

27.4

n.e.

n.e.

n.e.

n.e.

n.e.

150.0##

75.9

77.8

0.0

2.6

5.8

5.8S

1.5

300.0##P

118.1

86.8

0.2

1.9

4.3

3.8S

0.8

IIB

Solvent control1

100.0

100.0

0.1

2.9

1.0

1.0

0.0

Positive control4#

n.t.

71.3

0.0

1.8

14.0

13.5S

4.5

37.5

96.1

76.8

0.5***

2.6

0.0

0.0

0.0

75.0

91.6

89.0

0.1

2.1

3.0

3.0S

0.5

150.0

91.9

90.4

0.1

2.4

1.5

1.5

0.0

300.0

95.7

94.1

0.0

2.1

2.5

2.0S

0.0

Red characters in bold: Aberration frequency exceeding the lab’s historical control range data.

* Evaluation of 1000 metaphases per culture

** Inclusive cells carrying exchanges

*** Evaluation of 2000 metaphases per culture

#Evaluation of 50 metaphases per culture

##Evaluation of 200 metaphases per culture

n.e. Not evaluable due to unexpected cytotoxicity

n. t. Not tested

PPrecipitation occurred

SAberration frequency statistically significant higher than corresponding control values

1THF 0.5 % (v/v)

2EMS 900.0 µg/mL

3EMS 500.0 µg/mL

4CPA 1.4 µg/mL

Applicant's summary and conclusion

Conclusions:
ST 10 C 08 did not induce a toxicologically significant increase in the frequency of cells with aberrations or polyploid cells in either the presence or absence of a metabolising system. ST 10 C 08 is therefore considered to be non clastogenic to human lymphocytes in vitro.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, V79 cells of the Chinese Hamster were exposed to ST 10 C 08 diluted in tetrahydrofuran (THF) in three independent experiments. The following study design was performed:

 

Without S9-mix

With S9-mix

 

Exp. I

Exp. IIA

Exp. I, IIA, and IIB

Exposure period

4 h

18 h

4 h

Recovery

14 h

-

14 h

Preparation interval

18 h

18 h

18 h

 

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were scored for structural chromosome aberrations, except for the positive controls in Experiments I and II in the absence of S9 mix, where only 50 metaphases were scored.

The highest applied concentration (without S9 mix: 1200 µg/mL, approx. 5.0 mM; with S9 mix: 600 µg/mL, approx. 2.5 mM) was chosen with regard to the solubility properties of the test item in THF with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In Experiments I and IIA in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment I in the presence of S9 mix no cytotoxicity was observed. In Experiment IIA in the presence of S9 mix only at a concentration of 75.0 µg/mL cytotoxicity was observed. In Experiment IIB no cytotoxicity was observed up to the highest applied concentration.

In Experiment I no clastogenicity was observed at the evaluated concentrations either with or without metabolic activation.

In Experiment IIA in the presence of S9 mix cells treated with 75.0 µg/mL were not evaluable for cytogenetic damage due to unexpected cytotoxicity. The next higher concentration (150.0 µg/mL) induced a statistically significant increase in the number of aberrant cells (5.8 % aberrant cells excluding gaps). This value exceeded the laboratory’s historical control data range (0.0 – 4.0 aberrant cells excluding gaps).

To verify these observations a confirmatory experiment IIB was performed. The clastogenic effect of the test item at a concentration of 150.0 µg/mL was not reproduced and the test item is therefore considered as non-clastogenic.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

In the confirmatory experiment IIB treatment with 37.5 µg/mL led to a statistically significant increase in the number of endomitotic cells (0.5 %) compared to the solvent control. This effect was seen in only one of two experiments with the same study design. In addition, no dose-dependent increase in endomitotic cells was observed. Therefore, the single increased rate of endomitotic cells is regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Under the experimental conditions reported, ST 10 C 08 did not induce structural chromosome aberrations as determined by the chromosome aberration test inV79cells (Chinese hamster cell line)in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.