Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 & E.coli WP2uvrA.
- V79/HPRT Mammalian Cell Gene Mutation Assay with a Confirmatory Assay (OECD 476, GLP, K, rel. 2): non mutagenic up to cytotoxic concentrations.
- V79 chromosome aberration test (OECD 473, GLP, K, rel. 2): non clastogenic up to cytotoxic or precipitating concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-08-20 to 2008-09-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on 21th August 2007 / Signed on 15th October 2007)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene for S. thyphimurium and tryptophan gene for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from the livers of Sprague-Dawley rats treated with phenobarbitone/B-naphtoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Range-finding test: 15, 50, 150, 500, 1500 and 5000 μg/plate for all S. strains; 50, 150, 500, 1500 and 5000 μg/plate for E. coli.
Main test: 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test material was insoluble in water and dimethylsulphoxide at 50 mg/mL but was fully soluble in acetone at the same concentration in solubility checks performed in-house.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.2.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.2.1/1
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

OTHER EXAMINATIONS:
- Other: Observations of precipitate of the test substance

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 2006 & 2007).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per mL.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.
Evaluation criteria:
Dose-related increase in revertant frequency over the dose range tested and/or reproducible at one or more concentrations in at least one bacterial strain with or without metabolic activation.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met
Statistics:
Statistical methods, as recommended by the UKEMS can be used as an aid to evaluation. However statistical significance will not be the only determining factor for a positive response.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: the test material is not classified as a VOC.
- Water solubility: the test material was solubilised in acetone to improve solubility.
- Precipitation: A stringy, oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertants colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: the test material induced lowered revertant colony counts at the upper dose levels to TA100 but was non-toxic to WP2uvrA-. The test material formulation and S9-mix used in this experiment were both shown to be sterile. See table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2006 and 2007 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

Table 7.6.1/2: Test results: Preliminary toxicity test

Metabolic activation

Strain

Dose (µ/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

+

TA100

74

81

77

77

94

77

78

94

75

56

35

-

64

64

70

66

85

72

62

63

65

36

20

+

WP2

uvrA-

35

25

34

26

31

26

36

46

22

26

25

-

30

35

31

34

28

25

34

32

41

26

22

Conclusions:
ST 10 C 08 is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA- were exposed to ST 10 C 08 diluted in acetone both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 15 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day using a modified dose range, 50 to 5000 µg/plate based on the results of the range-finding test.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ST 10 C 08 caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A stringy, oily precipitate, was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, ST 10 C 08 is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

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".
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
Target gene:
Not applicable
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.
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).
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.

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

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-12-09 to 2009-05-26
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 minor restrictions: adequate toxicity was not achieved in all exposure groups.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
adequate toxicity not achieve in all exposure groups
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
adequate toxicity not achieve in all exposure groups
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
OECD GLP (inspected from 15 to 29, October 2008 / Signed on 30, March 2009)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM supplemented with 10 % foetal calf serum and 1 % neomycin. The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, before freezing the level of spontaneous mutants was depressed by treatment with HA medium
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
- Pre test: 18.8 to 2400.0 µg/mL
- Experiment I: without S9-mix: 0.25, 0.5, 1.0, 2.0, 3.0, 4.0 µg/mL / with S9-mix: 8.0, 16.0, 24.0, 40.0, 64.0, 72.0 µg/mL
- Experiment II: without S9-mix: 0.6, 1.3, 2.5, 5.0, 10.0, 20.0 µg/mL / with S9-mix: 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: test material not soluble in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.150 mg/mL (1.2 mM) in experiment 1; 0.075 mg/mL (0.6 mM) in experiment 2; dissolved in nutrient medium
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1.1 µg/mL (4.3 µM); dissolved in DMSO
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 4 h in Experiment I, 4 h with S9-mix and 24 hours without S9-mix in Experiment II
- Expression time (cells in growth medium): approx. 7 days
- Selection time (if incubation with a selection agent): 8 days, at 37 °C in a humidified atmosphere with 4.5 % CO2.
- Fixation time (start of exposure up to fixation or harvest of cells): approx. 15 days

SELECTION AGENT (mutation assays): thioguanine (6TG), 11 µg/mL

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (survival)

OTHER:
Determination of: cloning efficiency (Survival), cell density % of control, cells survived (after plating in TG containing medium) and induction factor
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
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 effect, a pH of 7.4 was determined in the solvent control and the maximum concentration of the pre-experiment without metabolic activation.
- Effects of osmolality: No effect, determined in the solvent control (360 mOsm) and the maximum concentration (345 mOsm) of the pre-experiment without metabolic activation:
- Evaporation from medium: the test material is not a VOC
- Water solubility: the test material was diluted in THF to improve its solubility
- Precipitation: The test item formed a clear phase separation in culture medium at 80 and 100 μg/mL
- Other confounding effects:

COMPARISON WITH HISTORICAL CONTROL DATA: In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 12.0 up to 23.6 mutants per 10E6 cells; these values are in the range of historical solvent control data of the lab.
EMS (150 μg/mL in experiment I and 75 μg/mL in experiment II) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies, both were in the range of historical positive control data of the lab.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Relevant cytotoxic effects as indicated by a relative cloning efficiency of less than 50 % in both parallel cultures occurred at 4.0 µg/mL in the first experiment without metabolic activation. In the second experiment with metabolic activation virtually no cells survived treatment at 80 µg/mL. No cytotoxic effects at all were noted at the next higher concentration of 100 µg/mL. This unusual dose response is based on solubility problems. The test item formed a clear phase separation in culture medium at 80 and 100 µg/mL. Isolated cytotoxic effects may occur at the limit of solubility based on super-saturation of the medium at the beginning of treatment. The large difference in the tolerated dose with and without metabolic activation indicates protein binding effects of the test item. The protein concentration is considerably higher in the experimental part with metabolic activation due to the S9
added. Protein binding effects are also indicated by the higher tolerated doses during long term treatment for 24h. Again, the protein concentration in the medium is higher due to 15% horse serum added. Protein binding is also the most likely reason for the deviation in cytotoxicity following 24 hours of treatment in the pre-experiment and the second experiment without metabolic activation. Strong toxic effects were noted in the pre-experiment at 18.8 µg/mL whereas no relevant cytotoxic effects occurred in the second experiment at 20 µg/mL. Minor changes in cell density may lead to variations of the free concentration of a
protein binding test item.

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The threshold of three times the mutation frequency of the corresponding solvent control was not reached at any of the test points.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

Conclusions:
ST 10 C 08 did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.
Executive summary:

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, V79 cells of the Chinese hamster were exposed to ST 10 C 08 diluted in tetrahydrofuran (THF), in duplicate in the presence and absence of metabolic activation (S9-mix).

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Experiment I had to be terminated prematurely due to unexpectedly severe toxicity. A repeat experiment was performed under identical general conditions but lower concentrations. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration of the test item in the pre-experiment was 2400 μg/mL equal to approximately 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and the solubility of the test item. The cell cultures were evaluated at the following concentrations:

Experiment I: without S9 mix: 0.5; 1.0; 2.0; 3.0; and 4.0 μg/mL; with S9 mix: 16.0; 24.0; 40.0; 64.0; and 72.0 μg/mL.

Experiment II: without S9 mix: 1.3; 2.5; 5.0; 10.0; and 20.0 μg/mL; with S9 mix: 10.0; 20.0; 40.0; 60.0; and 100.0 μg/mL.

Phase separation of ST 10 C 08 was observed at the two highest concentrations of the second experiment.

Relevant cytotoxic effects as indicated by a relative cloning efficiency of less than 50 % in both parallel cultures occurred at 4.0 μg/mL in the first experiment without metabolic activation. In the second experiment with metabolic activation virtually no cells survived treatment at 80 μg/mL. No cytotoxic effects at all were noted at the next higher concentration of 100 μg/mL. This unusual dose response is based on solubility problems. ST 10 C 08 formed a clear phase separation in culture medium at 80 and 100 μg/mL. Isolated cytotoxic effects may occur at the limit of solubility based on super-saturation of the medium at the beginning of treatment. The large difference in the tolerated dose with and without metabolic activation indicates protein binding effects of the test item. The protein concentration is considerably higher in the experimental part with metabolic activation due to the S9 added. Protein binding effects are also indicated by the higher tolerated doses during long term treatment for 24h. Again, the protein concentration in the medium is higher due to 15% horse serum added. Protein binding is also the most likely reason for the deviation in cytotoxicity following 24 hours of treatment in the pre-experiment and the second experiment without metabolic activation. Strong toxic effects were noted in the pre-experiment at 18.8 μg/mL whereas no relevant cytotoxic effects occurred in the second experiment at 20 μg/mL. Minor changes in cell density may lead to variations of the free concentration of a protein binding test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

 

Under the test conditions, ST 10 C 08 did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

This study is considered as acceptable and satisfies the requirement for mammalian gene mutation endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Safepharm, 2008

Ames Test

(OECD 471)

K, rel. 2

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100,

WP2 uvrA

-S9

+S9

Up to 5000 µg/plate

-S9 : non mutagenic

+S9 : non mutagenic

2

 

Harlan, 2009

V79/HPRT test (OECD 476)

K, rel. 2

Gene mutation

Chinese hamster V79 cells

-S9

+S9

Up to cytotoxic concentrations

-S9 : non mutagenic

+S9 : non mutagenic

3

 

Harlan, 2009

V79 CAT

(OECD 473)

K, rel. 2

Chromosomal aberration

Chinese hamster V79 cells

 

-S9

+S9

Up to cytotoxic or precipitating concentration

-S9 : non clastogenic

+S9 : non clastogenic

Gene mutation Assays (Tests n° 1 -2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 (See Table 1). The substance does not induce gene mutations in bacteria under the test conditions whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in Chinese hamster V79 cells (V79/HPRT test)(Test n°3). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments. ST 10 C 08 does not induce forward mutations at the HPRT locus in V79 chinese hamster cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore the substance is considered as negative for inducing gene mutations at the HPRT locus in V79 chinese hamster cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in Chinese hamster V79 cells, which measures the potential of a substance to increase the incidence of structural chromosome aberrations in V79 cells.

None of the dose levels up to the cytotoxicity limit or the precipitating limit with ST 10 C 08, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. ST 10 C 08 does not induce structural aberrations in the chromosomes of Chinese hamster V79 cells under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Therefore the substance is considered as negative for inducing chromosomal mutations in Chinese hamster V79 cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the CLp and the GHS.