Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the test and read across in vitro genotoxicity studies, the test substance is considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 23, 1990 to May 10, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine: hisC3076, hisD3052 pKM101, hisG46, hisG46 pKM101

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats

Test concentrations with justification for top dose:

Preliminary toxicity test:
1.0, 3.3, 10, 33.3, 100, 333, 1000, 3330, 5000 µg/plate

Experiment 1
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate

Experiment 2
with S9-mix
0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
without S9-mix
0.1, 0.33, 1.0, 3.3, 10.0 µg/plate

Vehicle / solvent:

Dimethylsulphoxide (DMSO) of spectroscopic quality

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA1535), 9-aminoacridine (TA1537), daunomycine (TA98), methylmethanesulfonate (TA100)

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all 4)

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: until 10^9 cells/mL had been obtained
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: Five different doses of the test substance have been tested in triplicate in each strain. An independentrepeat of the experiment was performed.

NUMBER OF CELLS EVALUATED: All colonies were counted.

DETERMINATION OF CYTOTOXICITY
Method: A preliminary toxicity test was performed with TA100 (with and without S9-mix), 9 concentrations were tested in duplicate. The survival of the TA100 culture was determined by comparing the number of colonies on the plate + test substance with those onthe solvent control plate.

Evaluation criteria:

An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory
historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times
the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding
test with strain TA100 or should extend to 5 mg/plate (active ingredient).

A test substance was considered negative (not mutagenic) i n the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance was considered positive (mutagenic)in the Ames test if :
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent
control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two
concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Statistics:

Not reported

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in preliminary test)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No data

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100
was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Conclusions:

Under the study conditions, the test substance was considered to be non mutagenic in the Ames test.

Executive summary:

A study was conducted to evaluate the mutagenic potential of the test substance, C12 -18 DAQ (76.4% active in hydroalcoholic solution) in the Ames test, according to the OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. In the study S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 strains were used and S9 liver mix obtained from Aroclor 1254 induced male Wistar or Sprague-Dawley rats was used as metabolic activating system. A preliminary toxicity study with TA100 showed that survival without S9-mix was not or only slightly reduced up to test substance concentration of 10.0 µg/plate and eliminated at and above 33.3 µg/plate. In the presence of S9-mix the survival of strain TA100 was slightly reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. Based on these data, the test substance was tested up to a concentration of 10.0 µg/plate in the absence of S9-mix and up to 33.3 µg/plate in the presence of S9-mix. Experiment 1: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. Experiment 2: (a) with S9-mix - 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate (b) without S9-mix - 0.1, 0.33, 1.0, 3.3, 10.0 µg/plate. The negative and strain-specific positive control values fell within the testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. Under the study conditions, the test substance was considered to be non mutagenic in the Ames test (Scheres, 1990).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 10, 1995 to March 06, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

cultured peripheral human lymphocytes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's, The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (OOC) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer, The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C).

Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.02 mg MgC12.6H20; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 umol HEPES. The above solution was filter (0.22 pm)-sterilized. To 0.5 ml S9-mix components 0.5 ml W-fraction (batch 95.7) was added (50% (v/v) S9-fraction). Metabolic activation was achieved by adding 0.2 ml liver S9-mix to 5.3 ml exposition medium (4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) Phytohaemagglutinin). The concentration of the S9-fraction in the exposition medium was 1.8% (v/v).

Test concentrations with justification for top dose:

First experiment: (a) Without S9-mix: 3-4.2 and 5.6 µg/mL (24 h) and 5.6 µg/mL (48h) (b) With S9-mix: 10-18 and 24 µg/mL (24 h) and 18 µg/mL (48 h)
Second experiment : (a) Without S9-mix: 3-4.2 and 7.5 µg/mL (24h) (b) With S9-mix: 3-18 and 24 µg/mL (24h)

Vehicle / solvent:

- DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix

Details on test system and experimental conditions:

Organism/cell type: Cultured peripheral human lymphocytes
Metabolic activation system: S9 mix: Aroclor-1254 induced rat liver SS-mix from adult male Wistar rats, from Charles River Wiga, Sulzfeld, Germany.

CELL CULTURE
Blood samples              
Blood samples were taken from a healthy adult male volunteer by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25°C. Within 4 h after blood collection lymphocyt cultures were started.

F10 complete culture medium       
F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat­ inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/1) and 30 U/ml heparin.

Cell culture conditions       
Whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). Per culture (5 ml F10 complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added.

Environmental conditions       
All incubations were carried out in a humid atmosphere (80-95%) containing 5% co2 in air in the dark at 37°C. The temperature, humidity and CO2- percentage were monitored during the experiment.

Negative control:
The vehicle of the test article, being dimethylsulphoxide (DMSO)

Positive controls:
Solvent for positive controls
Hank's Balanced Salt Solution (HBSS) without calcium and magnesium

Without metabolic activation (-S9-mix):
Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, U.S.A.) was used as a direct acting mutagen at a final concentration of 0.2 ug/ml (solvent: HBSS) for a 24 h treatment period and 0.1 ug/ml for a 48 h treatment period.

With metabolic activation (+S9-mix):
Cyclophosphamide (CP; CAS no. 50-18-O. Endoxan-Asta, Asta-Werke, F.R.G.) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 15 ug/ml (solvent: HBSS) for a 3 h treatment period (24 h fixation time).

Rationale for test conditions:

Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes. In combination with a mammalian metabolizing system (S9-mix) also indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, could be tested for clastogenic effects in vitro. Following treatment, cell division was arrested in the metaphase stage of the cell cycle by addition of the spindle poison colchicine. Structural chromosome changes such as breaks, gaps, minutes, dicentrics and exchange figures were examined microscopically in cultures treated with the test substance and the results were compared with those of the control (vehicle-treated) cultures. Chromosome aberrations were generally evaluated in the first post-treatment mitosis. The appearance of the first post-treatment mitosis could be considerably delayed, due to toxic insult of the cells. Therefore, cells were harvested at 24 hand 48 h after beginning of treatment to cover the interval in which maximum aberration frequency was expected. A test article which induced a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent.

Evaluation criteria:

Test was considered as acceptable, if it met the following criteria:
1. The Numbers of chromosome aberrations found in the solvent control cultures should reasonable be within the laboratory historical control.
2. The positive control substance should produce a statistically significant increase in the number of cells with chromosome aberrations
3. A homogeneous response between the replicate cultures is observed

Statistics:

Chi square test for the dose related statistical significance

Key result

Species / strain:

other: cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 24 µg/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- In the first experiment an increase in the number of aberrant cells was found, only at the 24 h fixation time in the presence of S9-mix and at one concentration only (24 µg/mL). 
- Since the type of aberrations abserved were only breaks, the increase was not observed at any other concentration and not confirmed in the second experiment, this finding was considered not to be of biological significance. 
- Next to this the number of cells with aberrations were just at the historical control data range.

Results

Dose range finding test

In the dose range finding test blood cultures were treated with 3, 10, 33, 100 and 333 µg/ml culture medium with and without S9-mix. At a concentration of 333 µg/ml test substance precipitated in the culture medium. Therefore, a concentration of 333 µg/ml was used as the highest concentration of test substance.

Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Experiment 1

Without S9-mix: (a) 3, 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (24 h fixation time) (b) 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (48 h fixation time)

With S9-mix: (a) 3, 10, 13, 18, 24 and 33 µg/ml culture medium (24 h fixation time) (b) 10, 13, 18, 24 and 33 µg/ml culture medium (48 h fixation time)

Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:

Without S9-mix: 3, 4.2 and 5.6 µg/ml cultuie medium (24 h fixation time), 5.6 µg/ml (48 h fixation time)

With S9- mix: 10, 18 and 24 µg/ml culture medium (24 h fixation time), 18 µg/ml (48 h fixation time)

The data of the dose range finding test and the first cytogenetic assay were used to determine the dose levels for the second cytogenetic assay.

Experiment 2

Without S9-mix: 1, 3, 4.2, 5.6 and 7.5 ug/ml culture medium (24 h fixation time)

With S9-mix: 3, 10, 13, 18, 21 and 24 µg/ml culture medium (24 h fixation time)

Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:

Without S9-mix: 3, 4.2 and 7 .5 µg/ml culture medium (24 h fixation time)

With S9-mix: 3, 18 and 24 µg/ml culture medium (24 h fi xati on time)

Cytogenetic assay

The ability of test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in two independent experiments. The results of duplicate cultures are indicated by A and B. The scores for the numbers of aberrant cells (inclusive and exclusive gaps) and the numbers of the various types of chromosome aberrations at the various concentrations of the test substance are presented. The criteria according to which the aberrations were classified are outlined. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps are included and excluded. Since, the types of aberrations observed were only breaks, the increase is not dose related, experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, the increase is not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {i.e.1.0 ± 3.3 (mean± three times the standard deviation) aberrant cells per 100 metaphases (without S9-mix; gaps excluded) and 0.8 ± 2.7 aberrant cells per 100 metaphases (with S9-mix; gaps excluded)}. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Finally, it is concluded that this test should be considered valid and that test substance is not clastogenic under the experimental conditions of this test.

Table 1. Table for Cytogenetic In-Vitro-Test: Chromosomal Analysis
	positive	Dose in µg/ml
state mean and standard deviations below	control
Experiment 1
Without S9-mix, 24 h fixation		control	3	4.2	5.6
Mitotic index	64	100	82	73	47
Total aberrations with gaps:	61	1	0	4	0
Total aberrations without gaps:	63	1	0	2	0
With S9-mix, 24 h fixation	pos. control	control	10	18	24
Mitotic index	59	100	81	58	41
Total aberrations with gaps:	53	1	6	0	9
Total aberrations without gaps:	52	0	4	0	9
Without S9-mix, 48 h fixation	pos.	control	5.6
Mitotic index	78	100	52
Total aberrations with gaps:	52	1	5
Total aberrations without gaps:	52	0	3
With S9-mix, 48 h fixation	pos.	control	18
Mitotic index	n.a.	100	49
Total aberrations with gaps:	na	1	6
Total aberrations without gaps:	na	1	4
Experiment 2
Without S9-mix, 24 h fixation	pos.	control	3	4.2	7.5
Mitotic index	58	100	83	61	45
Total aberrations with gaps:	33	2	2	2	8
Total aberrations without gaps:	33	1	1	2	4
With S9-mix, 24 h fixation	pos.	control	3	18	24
Mitotic index	51	100	79	61	49
Total aberrations with gaps:	36	3	4	1	5
Total aberrations without gaps:	36	3	2	1	3
Positve controls: the numbers are based on 150 instead 200 cells evaluated.

Conclusions:

Under the study conditions, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation.

Executive summary:

A study was conducted to determine the clastogenic potential of the test substance, DDAC (51.3% active) in cultured human lymphocytes, according to OECD Guideline 473 and EU method B.10, in compliance with GLP. After a preliminary toxicity test, test substance was tested in two independent experiments, with and without a metabolic activation system, the S9- mix. In the absence of S9 -mix test substance was tested up to 5.6 µg/mL for a 24 and 48 h fixation time in the first experiment. In the second experiment test substance was tested up to 7.5 µg/mL for a 24 h fixation time. In the presence of S9 -mix test substance was tested up to 24 µg/mL for 24 h fixation time and up to 18 µg/mL for a 48 h fixation time in the first experiment. In the second experiment it was tested up to 24 µg/mL for a 24 h fixation time. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since, the types of aberrations observed were only breaks, the increase was not dose related. Experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, therefore the increase was not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation (Van de waart, 1996).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From November 10, 1995 to March 06, 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River Wiga, Sulzfeld, Germany. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP's, The rats were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (OOC) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer, The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C).

Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.02 mg MgC12.6H20; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 umol HEPES. The above solution was filter (0.22 pm)-sterilized. To 0.5 ml S9-mix components 0.5 ml W-fraction (batch 95.7) was added (50% (v/v) S9-fraction). Metabolic activation was achieved by adding 0.2 ml liver S9-mix to 5.3 ml exposition medium (4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) Phytohaemagglutinin). The concentration of the S9-fraction in the exposition medium was 1.8% (v/v).

Test concentrations with justification for top dose:

First experiment: (a) Without S9-mix: 3-4.2 and 5.6 µg/mL (24 h) and 5.6 µg/mL (48h) (b) With S9-mix: 10-18 and 24 µg/mL (24 h) and 18 µg/mL (48 h)
Second experiment : (a) Without S9-mix: 3-4.2 and 7.5 µg/mL (24h) (b) With S9-mix: 3-18 and 24 µg/mL (24h)

Vehicle / solvent:

- DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix

Details on test system and experimental conditions:

Organism/cell type: Cultured peripheral human lymphocytes
Metabolic activation system: S9 mix: Aroclor-1254 induced rat liver SS-mix from adult male Wistar rats, from Charles River Wiga, Sulzfeld, Germany.

CELL CULTURE
Blood samples              
Blood samples were taken from a healthy adult male volunteer by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25°C. Within 4 h after blood collection lymphocyt cultures were started.

F10 complete culture medium       
F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat­ inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/1) and 30 U/ml heparin.

Cell culture conditions       
Whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). Per culture (5 ml F10 complete culture medium and 0.4 ml whole blood) 0.1 ml (9 mg/ml) Phytohaemagglutinin was added.

Environmental conditions       
All incubations were carried out in a humid atmosphere (80-95%) containing 5% co2 in air in the dark at 37°C. The temperature, humidity and CO2- percentage were monitored during the experiment.

Negative control:
The vehicle of the test article, being dimethylsulphoxide (DMSO)

Positive controls:
Solvent for positive controls
Hank's Balanced Salt Solution (HBSS) without calcium and magnesium

Without metabolic activation (-S9-mix):
Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, U.S.A.) was used as a direct acting mutagen at a final concentration of 0.2 ug/ml (solvent: HBSS) for a
24 h treatment period and 0.1 ug/ml for a 48 h treatment period.

With metabolic activation (+S9-mix):
Cyclophosphamide (CP; CAS no. 50-18-O. Endoxan-Asta, Asta-Werke, F.R.G.) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 15 ug/ml (solvent: HBSS) for a 3 h treatment period (24 h fixation time).

Rationale for test conditions:

Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes. In combination with a mammalian metabolizing system (S9-mix) also indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, could be tested for clastogenic effects in vitro. Following treatment, cell division was arrested in the metaphase stage of the cell cycle by addition of the spindle poison colchicine. Structural chromosome changes such as breaks, gaps, minutes, dicentrics and exchange figures were examined microscopically in cultures treated with the test substance and the results were compared with those of the control (vehicle-treated) cultures. Chromosome aberrations were generally evaluated in the first post-treatment mitosis. The appearance of the first post-treatment mitosis could be considerably delayed, due to toxic insult of the cells. Therefore, cells were harvested at 24 hand 48 h after beginning of treatment to cover the interval in which maximum aberration frequency was expected. A test article which induced a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent.

Evaluation criteria:

Test was considered as acceptable, if it met the following criteria:
1. The Numbers of chromosome aberrations found in the solvent control cultures should reasonable be within the laboratory historical control.
2. The positive control substance should produce a statistically significant increase in the number of cells with chromosome aberrations
3. A homogeneous response between the replicate cultures is observed

Statistics:

Chi square test for the dose related statistical significance

Key result

Species / strain:

other: cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 24 µg/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- In the first experiment an increase in the number of aberrant cells was found, only at the 24 h fixation time in the presence of S9-mix and at one concentration only (24 µg/mL). 
- Since the type of aberrations abserved were only breaks, the increase was not observed at any other concentration and not confirmed in the second experiment, this finding was considered not to be of biological significance. 
- Next to this the number of cells with aberrations were just at the historical control data range.

Results

Dose range finding test

In the dose range finding test blood cultures were treated with 3, 10, 33, 100 and 333 µg/ml culture medium with and without S9-mix. At a concentration of 333 µg/ml test substance precipitated in the culture medium. Therefore, a concentration of 333 µg/ml was used as the highest concentration of test substance.

Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Experiment 1

Without S9-mix: (a) 3, 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (24 h fixation time) (b) 4.2, 5.6, 7.5, 10 and 18 µg/ml culture medium (48 h fixation time)

With S9-mix: (a) 3, 10, 13, 18, 24 and 33 µg/ml culture medium (24 h fixation time) (b) 10, 13, 18, 24 and 33 µg/ml culture medium (48 h fixation time)

Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:

Without S9-mix: 3, 4.2 and 5.6 µg/ml cultuie medium (24 h fixation time), 5.6 µg/ml (48 h fixation time)

With S9- mix: 10, 18 and 24 µg/ml culture medium (24 h fixation time), 18 µg/ml (48 h fixation time)

The data of the dose range finding test and the first cytogenetic assay were used to determine the dose levels for the second cytogenetic assay.

Experiment 2

Without S9-mix: 1, 3, 4.2, 5.6 and 7.5 ug/ml culture medium (24 h fixation time)

With S9-mix: 3, 10, 13, 18, 21 and 24 µg/ml culture medium (24 h fixation time)

Based on the observations of the mitotic index of cultures (from blood of a healthy male donor) treated with various test substance concentrations or with the positive or negative control substances, the following doses were selected for scoring of chromosome aberrations:

Without S9-mix: 3, 4.2 and 7 .5 µg/ml culture medium (24 h fixation time)

With S9-mix: 3, 18 and 24 µg/ml culture medium (24 h fi xati on time)

Cytogenetic assay

The ability of test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in two independent experiments. The results of duplicate cultures are indicated by A and B. The scores for the numbers of aberrant cells (inclusive and exclusive gaps) and the numbers of the various types of chromosome aberrations at the various concentrations of the test substance are presented. The criteria according to which the aberrations were classified are outlined. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps are included and excluded. Since, the types of aberrations observed were only breaks, the increase is not dose related, experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, the increase is not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix test substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {i.e.1.0 ± 3.3 (mean± three times the standard deviation) aberrant cells per 100 metaphases (without S9-mix; gaps excluded) and 0.8 ± 2.7 aberrant cells per 100 metaphases (with S9-mix; gaps excluded)}. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Finally, it is concluded that this test should be considered valid and that test substance is not clastogenic under the experimental conditions of this test.

Table 1. Table for Cytogenetic In-Vitro-Test: Chromosomal Analysis
	positive	Dose in µg/ml
state mean and standard deviations below	control
Experiment 1
Without S9-mix, 24 h fixation		control	3	4.2	5.6
Mitotic index	64	100	82	73	47
Total aberrations with gaps:	61	1	0	4	0
Total aberrations without gaps:	63	1	0	2	0
With S9-mix, 24 h fixation	pos. control	control	10	18	24
Mitotic index	59	100	81	58	41
Total aberrations with gaps:	53	1	6	0	9
Total aberrations without gaps:	52	0	4	0	9
Without S9-mix, 48 h fixation	pos.	control	5.6
Mitotic index	78	100	52
Total aberrations with gaps:	52	1	5
Total aberrations without gaps:	52	0	3
With S9-mix, 48 h fixation	pos.	control	18
Mitotic index	n.a.	100	49
Total aberrations with gaps:	na	1	6
Total aberrations without gaps:	na	1	4
Experiment 2
Without S9-mix, 24 h fixation	pos.	control	3	4.2	7.5
Mitotic index	58	100	83	61	45
Total aberrations with gaps:	33	2	2	2	8
Total aberrations without gaps:	33	1	1	2	4
With S9-mix, 24 h fixation	pos.	control	3	18	24
Mitotic index	51	100	79	61	49
Total aberrations with gaps:	36	3	4	1	5
Total aberrations without gaps:	36	3	2	1	3
Positve controls: the numbers are based on 150 instead 200 cells evaluated.

Conclusions:

Based on the results of the read across study, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation.

Executive summary:

A study was conducted to determine the clastogenic potential of the read across substance, DDAC (51.3% active) in cultured human lymphocytes, according to OECD Guideline 473 and EU method B.10, in compliance with GLP. After a preliminary toxicity test, read across substance was tested in two independent experiments, with and without a metabolic activation system, the S9- mix. In the absence of S9 -mix read across substance was tested up to 5.6 µg/mL for a 24 and 48 h fixation time in the first experiment. In the second experiment read across substance was tested up to 7.5 µg/mL for a 24 h fixation time. In the presence of S9 -mix read across substance was tested up to 24 µg/mL for 24 h fixation time and up to 18 µg/mL for a 48 h fixation time in the first experiment. In the second experiment it was tested up to 24 µg/mL for a 24 h fixation time. In the presence of S9-mix (24 h fixation time), in experiment 1, a concentration of 24 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since, the types of aberrations observed were only breaks, the increase was not dose related. Experiment 2 did not confirm this result and moreover the number of cells with chromosome aberrations were just without the historical control data range, therefore the increase was not considered biologically relevant. All other concentrations tested in the presence of S9-mix at the 24 hand 48 h fixation period did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. In the absence of S9-mix read across substance did not induce a statistically and biologically significant increase in the number of cells with chromosome aberrations. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly (Van der waart, 1996). Based on the results of the read across study, the test substance was considered to be non clastogenic in human lymphocytes with and without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 27, 2001 to March 05, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Not applicable

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK (Thymidine kinase ) -/+, not able to grow in Trifluorothimidine medium

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, from Aroclor 1254 induced (500 mg/kg i.p.) rat livers. The final concentration of S9 fraction in the culture medium was 2%.

Test concentrations with justification for top dose:

Experiments without S9 mix:
0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL (1st experiment),
0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL (2nd experiment)
Experiments with S9 mix:
0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL (1st experiment),
0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL (2nd experiment).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

Cells
L5178Y cells are an established cell line recommended by international regulations for use in the in vitro mammalian cell gene mutation test. They have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. The average cell cycle time is 12-14 hours and the TK phenotypic expression time is 2 days. L5178Y cells, were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (77601, Marne-La-Vallée, France). The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethyl-sulfoxide (DMSO)) at -80°C or in liquid nitrogen. Each batch of frozen cells was purged of TK- mutants and checked for the absence of mycoplasma.

Metabolic activation system
The S9 mix consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Annapolis, MD 21401, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80°C, until use. The S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to culture medium.

Culture medium
RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10% v/v (RPMI 10) or 20% v/v (RPMI 20).

DURATION
- Exposure duration:
Without S9-mix: 1st experiment: 3 h; 2nd experiment: 24 h
With S9-mix: 1st experiment: 3 h; 2nd experiment: 3 h

NUMBER OF REPLICATIONS: Two plates/dose-level for test and four plates for control

NUMBER OF CELLS EVALUATED: 2000 cells/well

Rationale for test conditions:

For details on treatment of results, kindly refer to attached background material section of the IUCLID.

Evaluation criteria:

Acceptance criteria
This study was considered valid since the following criteria were fulfilled:
• the cloning efficiency of the vehicle controls were between 0.6-1.4 for CE0 and between 0.7-1.3 for CE2,
• the mutation frequency of the vehicle controls were between 60-250E-6
• the mutation frequency of the positive controls were higher than that of the vehicle controls (more than two fold) and consistent with our historical data.

Evaluation criteria
A reproducible noteworthy (at least two-fold) increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of the data obtained. Positive response observed only at high levels of cytotoxicity (survival lower than 10%) were not considered.

Statistics:

Not reported

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9-mix: 5.56 µg/mL at 3 h treatment; 0.56 µg/mL at 24 h treatment; With S9-mix >5.56 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
-The test item was freely soluble in the vehicle (DMSO) at 380 mg/mL (expressed as active item). In the culture medium, the dose-level of 3800 µg/mL showed a marked emulsion. At this dose-level, the pH was approximately 7.9 (7.4 for the vehicle control) and no increase in the osmolality due to the test item was noted. Consequently, with a treatment volume of 200 µL/20 mL (1% v/v) culture medium, the dose-levels for the preliminary toxicity test were: 7.6, 76, 380, 760, 1900 and 3800 µg/mL, both with and without S9.

-A slight to marked emulsion was observed at the end of the treatment period at dose-levels ≥ 760 µg/mL.

-Without S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 7.6 µg/mL (93-100% decrease in the cloning efficiency immediately after treatment (CE0) and in the relative survival (RS)).

-With S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 76 µg/mL (97-100% decrease in the CE0 and RS).

Results

Based on the preliminary toxicity test for the selection of the doses, the selected dose-levels for treatment were as follow : All the dose-levels were expressed as active substance, taking into account the active material content of 40.37%.

Without S9 mix
- First experiment: 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL
- Second Experiment: 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL

After 3 h treatment (first experiment), a slight to strong toxicity was showed by 39-69% decrease in the relative  total  growth (RTG)  at  dose- levels  >= 2.78 µg/mL and 96% decrease in the RS at 5.56 µg/mL. After 24 h treatment (second experiment), the test substance was moderately to strongly toxic at dose-levels >=0.56 µl/mL (as shown mainly by 57 -100% decrease in the RS). No noteworthy increase in the mutation frequency was induced both after 3 and 24 h treatments.

With S9 mix
-First experiment: 0.21, 0.62, 1.85, 5.56 16.7 and 50 µg/mL
-Second Experiment: 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL

The test substance was moderately to markedly toxic at dose-levels between 5 and 7.5 µg/mL. At higher dose-levels, the test substance was strongly to completely toxic. No noteworthy increase in the mutation frequencies was induced in both experiments.

The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria.  The study was therefore considered valid.

Table: First experiment without S9 mix, 3-hour treatment: mutagenicity results

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF 10-6	R
	40			11			74	18	4
0	24	0.7	100	18	1.0	100	79	12	5	121	1.0
	28			25			76	14	6
	38			26			74	16	6
0.07	25	0.8	121	14	0.9	88	79	13	4	105	0.9
	27			34			81	10	5
0.21	36	0.6	96	8	1.0	103	72	17	7	119	1.0
	32			30			79	12	5
0.62	35	0.6	93	11	1.0	102	73	16	7	137	1.1
	35			28			73	21	2
1.85	29	0.7	109	6	1.2	118	80	6	6	84	0.7
	30			24			78	12	6
2.78	24	0.8	124	7	1.0	100	81	8	7	68	0.6
	26			33			87	6	3
5.56	31	0.8	122	32	0.7	72	71	17	10	179	1.5
	20			30			78	7	11
MMS	44	0.6	86	34	0.5	49	49	16	31	658	5.4
25 µg/ml	32			55			53	21	23

0: vehicle control ( DMSO )

MMS:methylmethanesulfonate

LC: large colonies

CE₀and CE₂: cloning efficiency

SC: small colonies

RCE₀and RCE₂: relative cloning efficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

Table : First experiment without S9 mix, 3-hour treatment: cell growth during expression time

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	2.7	2.0	11.0	2.0	4.7	5.5	2.4	13.0
0									100	100
	3.6	2.0	7.5	2.0	6.3	3.8	3.2	11.8
	3.1	2.0	7.1	2.0	6.0	3.5	3.0	10.5
0.07									76	67
	3.7	2.0	6.2	2.0	5.4	3.1	2.7	8.4
	3.9	2.0	5.6	2.0	5.3	2.8	2.7	7.4
0.21									72	74
	3.5	2.0	7.1	2.0	5.9	3.6	2.9	10.4
	4.2	2.0	5.0	2.0	5.9	2.5	3.0	7.3
0.62									69	70
	3.9	2.0	6.9	2.0	5.7	3.4	2.9	9.8
	3.2	2.0	6.2	2.0	6.4	3.1	3.2	9.8
1.85									69	82
	3.1	2.0	5.3	2.0	5.6	2.6	2.8	7.3
	2.6	2.0	5.6	2.0	5.6	2.8	2.8	7.8
2.78									61	61
	2.3	2.0	5.3	2.0	5.6	2.7	2.8	7.4
	0.2	0.2 *	0.3	0.3 *	0.7	1.7	2.7	4.5
5.56									44	31
	0.1	0.1 *	0.1	0.1 *	0.4	2.0	3.1	6.3
	3.8	2.0	4.3	2.0	5.5	2.2	2.7	5.9
MMS									60	29
25 µg/ml	3.1	2.0	6.5	2.0	5.6	3.2	2.8	8.9

0: vehicle control ( DMSO )

conc.: cell concentration before being adjusted

adj.conc.:adjusted cell concentration when replating

SG: suspension growth

RSG: relative suspension growth

RTG: relative total growth

MMS: methylmethane sulfonate

*: cells were not replated as their density was < 2 x 10⁵/ml

Table: First experiment without S9 mix, 3-hour treatment: toxicity immediately after treatment

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	2.7
0		1.0	0.7	0.7	100
	3.6
	3.1
0.07		1.1	0.8	0.9	130
	3.7
	3.9
0.21		1.2	0.6	0.8	115
	3.5
	4.2
0.62		1.3	0.6	0.8	121
	3.9
	3.2
1.85		1.0	0.7	0.7	110
	3.1
	2.6
2.78		0.8	0.8	0.7	97
	2.3
	0.2
5.56		0.0	0.8	0.0	4
	0.1
	3.8
MMS		1.1	0.6	0.6	95
25 µg/ml	3.1

0: vehicle control (DMSO )

CE0: cloning efficiency immediately after treatment

RS: relative survival

MMS: methylmethane sulfonate

Table : Second experiment without S9 mix, 24-hour treatment: mutagenicity results

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF10-6	R
	35			34			85	6	5
0	34	0.7	100	36	0.7	100	84	8	4	124	1.0
	32			32			80	12	4
	28			32			77	11	8
0.06	28	0.6	95	28	0.8	124	82	10	5	85	0.7
	40			24			85	7	5
0.19	34	0.7	101	31	0.7	103	82	8	7	81	0.7
	30			34			90	6	0
0.56	36	0.6	91	31	0.7	103	84	7	5	64	0.5
	35			34			92	3	1
1.67	35	0.4	54	23	0.7	107	85	7	4	74	0.6
	71			39			88	1	7
2.5	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
5	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
MMS	29	0.8	122	36	0.6	97	56	16	24	394	3.2
5 µg/ml	22			33			60	23	15

0: vehicle control ( DMSO )

MMS: methylmethanesulfonate           

LC: large colonies

CE₀and CE₂: cloning efficiency

SC: small colonies

RCE₀and RCE₂: relative cloning efficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

-: not evaluated due to severe toxicity observed

Table : Second experiment without S9 mix, 24-hour treatment: cell growthduring expressiontime

 

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	5.8	2.0	6.8	2.0	6.3	3.4	3.1	10.5
0									100	100
	8.4	2.0	8.4	2.0	5.5	4.2	2.8	11.6
	6.2	2.0	5.5	2.0	5.7	2.8	2.9	7.8
0.06									84	104
	5.2	2.0	7.1	2.0	6.1	3.6	3.0	10.7
	5.5	2.0	6.2	2.0	5.0	3.1	2.5	7.7
0.19									79	82
	5.4	2.0	6.8	2.0	5.8	3.4	2.9	9.9
	4.1	2.0	7.4	2.0	6.0	3.7	3.0	10.9
0.56									101	104
	2.6	2.0	6.3	2.0	7.4	3.1	3.7	11.5
	0.0	0.0 *	0.2	0.2 *	0.4	4.8	2.1	9.8
1.67									59	63
	0.1	0.1 *	0.1	0.1 *	0.3	0.7	4.7	3.3
	-	-	-	-	-	-	-	-
2.5									-	-
	-	-	-	-	-	-	-	-
	-	-	-	-	-	-	-	-
5									-	-
	-	-	-	-	-	-	-	-
	7.8	2.0	7.7	2.0	4.8	3.9	2.4	9.2
MMS									82	80
5 µg/ml	7.4	2.0	7.0	2.0	5.1	3.5	2.6	8.9

0:vehiclecontrol(DMSO)

conc.: cell concentration before being adjusted

adj.conc.:adjusted cell concentration when replating

SG: suspension growth

RSG: relative suspension growth

RTG: relative total growth

MMS: methylmethane sulfonate

*: cells were not replated as their density was < 2 x 10⁵/ml

-: not evaluated due to severe toxicity observed

Table : Second experiment without S9 mix, 24-hour treatment: toxicity immediately after treatment

 

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	5.8
0		1.0	0.7	0.7	100
	8.4
	6.2
0.06		0.8	0.6	0.5	76
	5.2
	5.5
0.19		0.8	0.7	0.5	77
	5.4
	4.1
0.56		0.5	0.6	0.3	43
	2.6
	0.0
1.67		0.0	0.4	0.0	0
	0.1
	-
2.5		-	-	-	-
	-
	-
5		-	-	-	-
	-
	7.8
MMS		1.1	0.8	0.9	130
5 µg/ml	7.4

0: vehicle control ( DMSO )

CE0: cloning efficiency immediately after treatment

RS: relative survival

MMS: methylmethane sulfonate

-: not evaluated due to severe toxicity observed

Table: First experiment with S9 mix, 3-hour treatment: mutagenicity results

 

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF10-6	R
	8			10			77	11	8
0	6	1.4	100	14	1.3	100	82	9	5	75	1.0
	17			21			80	11	5
	12			4			78	9	9
0.21	5	2.1	151	5	1.8	139	83	9	4	46	0.6
	2			6			80	11	5
0.62	5	1.7	123	11	1.4	112	77	15	4	52	0.7
	8			8			88	6	2
1.85	3	2.1	151	4	1.6	127	76	9	11	56	0.7
	4			10			84	2	10
5.56	13	1.4	106	16	1.0	80	85	2	9	45	0.6
	6			21			90	2	4
16.7	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
50	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
CPA	13	1.1	78	31	0.7	56	41	27	29	491	6.6
3 µg/ml	22			30			54	13	30

0: vehicle control ( DMSO )

CPA:cyclophosphamide          

LC: large colonies

CE₀and CE₂:cloningefficiency

SC: small colonies

RCE₀and RCE₂: relative cloningefficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

-: not evaluated due to severe toxicity observed

Table : First experiment with S9 mix, 3-hour treatment: cell growth during expression time

 

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	3.8	2.0	8.0	2.0	7.3	4.0	3.6	14.5
0									100	100
	4.4	2.0	6.9	2.0	7.2	3.5	3.6	12.4
	4.2	2.0	7.2	2.0	7.5	3.6	3.7	13.4
0.21									113	157
	4.1	2.0	8.5	2.0	8.1	4.2	4.0	17.0
	4.3	2.0	9.1	2.0	5.4	4.5	2.7	12.2
0.62									103	116
	3.6	2.0	9.2	2.0	6.8	4.6	3.4	15.5
	3.8	2.0	6.1	2.0	5.6	3.0	2.8	8.5
1.85									67	85
	4.8	2.0	5.1	2.0	7.5	2.5	3.8	9.5
	3.1	2.0	6.5	2.0	3.1	3.2	1.5	5.0
5.56									38	31
	3.4	2.0	4.3	2.0	5.0	2.2	2.5	5.4
	0.0	-	-	-	-	-	-	-
16.7									-	-
	0.0	-	-	-	-	-	-	-
	0.0	-	-	-	-	-	-	-
50									-	-
	0.0	-	-	-	-	-	-	-
	3.8	2.0	6.1	2.0	6.1	3.1	3.0	9.2
CPA									82	46
3 µg/ml	3.9	2.0	5.6	2.0	9.3	2.8	4.6	13.0

0: vehicle control ( DMSO )

conc.: cell concentration before being adjusted

adj.conc.:adjustedcellconcentrationwhenreplating SG: suspensiongrowth

RSG: relative suspension growth RTG: relative total growth

CPA: cyclophosphamide

-: not evaluated due to severe toxicity observed

Table : First experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment

 

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	3.8
0		1.0	1.4	1.4	100
	4.4
	4.2
0.21		1.0	2.1	2.1	153
	4.1
	4.3
0.62		1.0	1.7	1.6	119
	3.6
	3.8
1.85		1.0	2.1	2.2	158
	4.8
	3.1
5.56		0.8	1.4	1.1	83
	3.4
	0.0
16.7		0.0	-	-	-
	0.0
	0.0
50		0.0	-	-	-
	0.0
	3.8
CPA		0.9	1.1	1.0	73
3 µg/ml	3.9

0: vehicle control ( DMSO )

CE0: cloning efficiency immediately after treatment RS: relative survival

CPA: cyclophosphamide

-: not evaluated due to severe toxicity observed

Table : Second experiment with S9 mix, 3-hour treatment: mutagenicity results

 

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF 10-6	R
	24			12			82	9	5
0	30	0.8	100	21	1.1	100	82	9	5	67	1.0
	23			7			82	9	5
	30			25			85	5	6
0.19	30	0.8	96	10	1.4	122	80	9	9	60	0.9
	26			12			83	10	3
0.56	28	0.8	101	6	1.3	117	82	12	2	61	0.9
	25			18			82	9	5
1.67	17	0.9	110	6	1.4	127	83	9	4	67	1.0
	30			14			76	14	6
5	32	0.7	92	3	2.0	179	79	9	8	57	0.9
	27			5			74	14	9
	40			5			80	11	5
7.5		0.6	74		1.6	144				48	0.7
	35			10			85	6	5
10	49	0.4	53	26	0.7	64	91	5	0	26	0.4
	48			36			94	2	0
CPA	38	0.5	65	26	0.7	63	60	15	22	440	6.6
3 µg/ml	46			37			44	19	37

0: vehicle control ( DMSO )

CPA:cyclophosphamide          

LC: largecolonies

CE₀and CE₂:cloning efficiency

SC: small colonies

RCE₀and RCE₂: relative cloningefficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

Table : Second experiment with S9 mix, 3-hour treatment: cell growth during expression time

 

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	5.7	2.0	6.9	2.0	9.6	3.5	4.8	16.5
0									100	100
	5.6	2.0	7.0	2.0	7.9	3.5	4.0	13.7
	5.2	2.0	8.3	2.0	6.3	4.1	3.2	13.0
0.19									84	102
	5.1	2.0	7.5	2.0	6.6	3.8	3.3	12.4
	5.7	2.0	8.0	2.0	5.9	4.0	3.0	11.7
0.56									74	87
	6.5	2.0	5.4	2.0	8.0	2.7	4.0	10.7
	4.9	2.0	7.5	2.0	5.1	3.7	2.5	9.4
1.67									57	73
	6.5	2.0	5.3	2.0	6.0	2.6	3.0	7.8
	6.6	2.0	3.6	2.0	5.1	1.8	2.6	4.6
5									29	52
	5.5	2.0	5.2	2.0	3.3	2.6	1.6	4.2
	4.6	2.0	2.0	2.0 *	2.0	1.0	1.0	1.0
7.5									9	13
	5.1	2.0	2.4	2.0	2.7	1.2	1.3	1.6
	2.7	2.0	2.0	2.0 *	1.1	1.0	0.5	0.6
10									4	2
	2.4	2.0	1.4	1.4 *	1.1	0.7	0.8	0.5
	6.4	2.0	5.5	2.0	7.1	2.8	3.5	9.7
CPA									61	38
3 µg/ml	6.7	2.0	6.2	2.0	5.7	3.1	2.9	8.8

0: vehicle control ( DMSO )

conc.: cell concentration before being adjusted

adj.conc.:adjusted cell concentration when replating

SG: suspensiongrowth

RSG: relative suspension growth

RTG: relative total growth

CPA: cyclophosphamide

*: cells were not replated as their density was < 2 x 10⁵/ml

Table : Second experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment

  

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	5.7
0		1.0	0.8	0.8	100
	5.6
	5.2
0.19		0.9	0.8	0.7	87
	5.1
	5.7
0.56		1.1	0.8	0.9	109
	6.5
	4.9
1.67		1.0	0.9	0.9	111
	6.5
	6.6
5		1.1	0.7	0.8	98
	5.5
	4.6
7.5		0.9	0.6	0.5	63
	5.1
	2.7
10		0.4	0.4	0.2	24
	2.4
	6.4
CPA		1.2	0.5	0.6	75
3 µg/ml	6.7

0: vehicle control ( DMSO )

CE0: cloning efficiency immediately after treatment

RS: relative survival

CPA: cyclophosphamide

Table: Historical data without S9 mix - Mouse lymphoma assay

	3-hour treatment						24-hour treatment
assay No.	vehicle			MMS			vehicle			MMS
	CE0	CE2	MF	CE0	CE2	MF	CE0	CE2	MF	CE0	CE2	MF
1	1.2	0.9	84	0.8	0.5	682	0.6	0.8	118	0.4	0.6	539
2	0.9	0.7	82	0.9	0.5	528	0.6	0.8	128	0.5	0.5	603
3	1.1	0.9	139	1	0.6	467	0.7	0.9	242	0.6	0.6	601
4	-	-	-	-	-	-	0.7	0.7	78	0.5	0.5	475
5	0.7	0.8	124	0.8	0.4	499	0.7	0.7	126	0.5	0.6	479
6	0.9	0.7	117	0.7	0.5	424	0.6	0.8	143	0.4	0.5	755
7	0.9	0.8	108	0.7	0.5	431	0.7	0.8	72	1.1	0.6	426
8	0.6	0.7	126	0.6	0.4	612	0.6	0.7	161	0.4	0.5	990
9	1.0	0.8	130	1.1	0.5	605	-	-	-	-	-	-
10	1.1	1.1	61	1.1	0.5	861	0.7	1.1	87	1.1	0.7	675
11	1.2	0.9	75	0.9	0.5	458	0.6	0.7	125	0.5	0.4	1298
12	1.3	0.9	58	1.8	0.7	462	1.0	1.3	94	0.8	0.5	823
13	1.0	1.3	77	1.2	0.6	533	1.0	1.3	94	0.8	0.5	823
minimum	0.6	0.7	58	0.6	0.4	424	0.6	0.7	72	0.4	0.4	426
maximum	1.3	1.3	139	1.8	0.7	861	1.0	1.3	242	1.1	0.7	1298
mean	1.0	0.9	98	1.0	0.5	547	0.7	0.9	122	0.6	0.5	707

CE₀: cloning efficiency at day 0

CE²: cloning efficiency at day 2

MF: mutation frequency

MMS: methylmethane sulfonate (3-hour treatment: 25µg/ml ; 24-hour treatment: 5µg/ml)

- : not performed

Table: Historical data with S9 mix Mouse lymphoma assay

assay No.	vehicle			CPA
	CE0	CE2	MF	CE0	CE2	MF
1	0.9	0.8	83	0.5	0.4	1077
2	0.9	0.7	123	0.5	0.3	915
3	1.0	0.8	68	0.5	0.6	948
4	0.7	0.8	138	0.3	0.4	1177
5	0.7	1.0	80	0.5	0.4	1248
6	0.9	0.9	161	0.4	0.3	1200
7	0.7	0.8	186	0.4	0.4	958
8	0.8	0.7	137	0.4	0.3	1563
9	0.7	1.0	92	0.3	0.3	1200
10	0.6	1.0	136	0.4	0.5	643
11	0.9	0.8	246	0.7	0.8	618
12	0.6	0.8	199	0.4	0.4	1389
13	0.7	0.8	114	0.4	0.4	915
14	0.9	1.0	88	1.0	0.8	484
15	1.1	0.9	77	0.4	0.4	1077
16	0.7	0.8	86	0.4	0.7	675
17	0.8	0.7	113	0.5	0.5	754
18	0.7	0.9	91	0.5	0.4	1583
19	0.6	0.8	108	0.4	0.4	1126
20	1.2	1.0	79	1.4	0.7	544
21	0.7	1.2	72	0.6	0.8	636
22	0.9	1.2	86	0.7	0.9	721
23	1.0	0.8	108	0.6	0.3	1456
24	0.9	1.2	86	0.7	0.9	721
minimum	0.6	0.7	68	0.3	0.3	484
maximum	1.2	1.2	246	1.4	0.9	1583
mean	0.8	0.9	115	0.5	0.5	985

CE₀: cloning efficiency at day 0

CE₂: cloning efficiency at day 2

MF: mutation frequency

CPA: cyclophosphamide (3µg/ml)

- : not performed

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic in mouse lymphoma assay with and without metabolic activation.

Executive summary:

A study was conducted to determine the potential of the test substance, DDAC (40.37% active) to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells, according to OECD guideline 476 and EU method B17, in compliance with GLP. After a preliminary toxicity test, test substance was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5E+6 (3-hour treatment: preliminary toxicity test and all experiments except for the second experiment without S9 mix) or 0.15E+6 (24-hour treatment: second experiment without S9 mix) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. The test substance was dissolved in dimethylsulfoxide (DMSO). All the dose-levels were expressed as active substance, taking into account the active material content of 40.37%. The selected dose-levels for test substance treatment without S9 mix were as follows: (a) 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL, for the first experiment (b) 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL, for the second experiment. The selected dose-levels for test substance treatment with S9 mix were as follows: (a) 0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL, for the first experiment (b) 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL, for the second experiment. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking. The dose-levels for the positive controls were as follows: (1) without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL (3-hour treatment) or 5 µg/mL (24-hour treatment) (2) with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 µg/mL. Cytotoxicity was then determined using cloning efficiency (CE0) before expression of the mutant phenotype. Cell viability (using cloning efficiency CE2) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Other parameters such as cell concentration, relative survival (RS), relative suspension growth (RSG) and/or relative total growth (RTG) were also taken into consideration. In experiments without S9 mix. a slight to strong toxicity was induced, depending on the dose-levels and the treatment duration. No noteworthy increase in the mutation frequency was induced both experiments without S9 mix after the 3 and 24-hour treatments. In experiments with S9 mix, the test substance was moderately to strongly toxic, depending on the dose-levels. No noteworthy increase in the mutation frequency was induced in both experiments with S9 mix. The cloning efficiencies CE0 and CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Under the study conditions, the test substance was considered to be non-mutagenic in mouse lymphoma assay with and without metabolic activation (Haddouk, 2002).

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From November 27, 2001 to March 05, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Not applicable

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK (Thymidine kinase ) -/+, not able to grow in Trifluorothimidine medium

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, from Aroclor 1254 induced (500 mg/kg i.p.) rat livers. The final concentration of S9 fraction in the culture medium was 2%.

Test concentrations with justification for top dose:

Experiments without S9 mix:
0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL (1st experiment),
0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL (2nd experiment)
Experiments with S9 mix:
0.21, 0.62, 1.85, 5.56, 16.7 and 50 µg/mL (1st experiment),
0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL (2nd experiment).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

Cells
L5178Y cells are an established cell line recommended by international regulations for use in the in vitro mammalian cell gene mutation test. They have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. The average cell cycle time is 12-14 hours and the TK phenotypic expression time is 2 days. L5178Y cells, were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (77601, Marne-La-Vallée, France). The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethyl-sulfoxide (DMSO)) at -80°C or in liquid nitrogen. Each batch of frozen cells was purged of TK- mutants and checked for the absence of mycoplasma.

Metabolic activation system
The S9 mix consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Annapolis, MD 21401, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route. The S9 fraction was preserved in sterile tubes at -80°C, until use. The S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to culture medium.

Culture medium
RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10% v/v (RPMI 10) or 20% v/v (RPMI 20).

DURATION
- Exposure duration:
Without S9-mix: 1st experiment: 3 h; 2nd experiment: 24 h
With S9-mix: 1st experiment: 3 h; 2nd experiment: 3 h

NUMBER OF REPLICATIONS: Two plates/dose-level for test and four plates for control

NUMBER OF CELLS EVALUATED: 2000 cells/well

Rationale for test conditions:

For details on treatment of results, kindly refer to attached background material section of the IUCLID.

Evaluation criteria:

Acceptance criteria
This study was considered valid since the following criteria were fulfilled:
• the cloning efficiency of the vehicle controls were between 0.6-1.4 for CE0 and between 0.7-1.3 for CE2,
• the mutation frequency of the vehicle controls were between 60-250E-6
• the mutation frequency of the positive controls were higher than that of the vehicle controls (more than two fold) and consistent with our historical data.

Evaluation criteria
A reproducible noteworthy (at least two-fold) increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of the data obtained. Positive response observed only at high levels of cytotoxicity (survival lower than 10%) were not considered.

Statistics:

Not reported

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9-mix: 5.56 µg/mL at 3 h treatment; 0.56 µg/mL at 24 h treatment; With S9-mix >5.56 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
-The test item was freely soluble in the vehicle (DMSO) at 380 mg/mL (expressed as active item). In the culture medium, the dose-level of 3800 µg/mL showed a marked emulsion. At this dose-level, the pH was approximately 7.9 (7.4 for the vehicle control) and no increase in the osmolality due to the test item was noted. Consequently, with a treatment volume of 200 µL/20 mL (1% v/v) culture medium, the dose-levels for the preliminary toxicity test were: 7.6, 76, 380, 760, 1900 and 3800 µg/mL, both with and without S9.

-A slight to marked emulsion was observed at the end of the treatment period at dose-levels ≥ 760 µg/mL.

-Without S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 7.6 µg/mL (93-100% decrease in the cloning efficiency immediately after treatment (CE0) and in the relative survival (RS)).

-With S9 mix, the test item was markedly to strongly toxic at dose-levels ≥ 76 µg/mL (97-100% decrease in the CE0 and RS).

Results

Based on the preliminary toxicity test for the selection of the doses, the selected dose-levels for treatment were as follow : All the dose-levels were expressed as active substance, taking into account the active material content of 40.37%.

Without S9 mix
- First experiment: 0.07, 0.21, 0.62, 1.85, 2.78 and 5.56 µg/mL
- Second Experiment: 0.06, 0.19, 0.56, 1.67, 2.5 and 5 µg/mL

After 3 h treatment (first experiment), a slight to strong toxicity was showed by 39-69% decrease in the relative  total  growth (RTG)  at  dose- levels  >= 2.78 µg/mL and 96% decrease in the RS at 5.56 µg/mL. After 24 h treatment (second experiment), the test substance was moderately to strongly toxic at dose-levels >=0.56 µl/mL (as shown mainly by 57 -100% decrease in the RS). No noteworthy increase in the mutation frequency was induced both after 3 and 24 h treatments.

With S9 mix
-First experiment: 0.21, 0.62, 1.85, 5.56 16.7 and 50 µg/mL
-Second Experiment: 0.19, 0.56, 1.67, 5, 7.5 and 10 µg/mL

The test substance was moderately to markedly toxic at dose-levels between 5 and 7.5 µg/mL. At higher dose-levels, the test substance was strongly to completely toxic. No noteworthy increase in the mutation frequencies was induced in both experiments.

The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria.  The study was therefore considered valid.

Table: First experiment without S9 mix, 3-hour treatment: mutagenicity results

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF 10-6	R
	40			11			74	18	4
0	24	0.7	100	18	1.0	100	79	12	5	121	1.0
	28			25			76	14	6
	38			26			74	16	6
0.07	25	0.8	121	14	0.9	88	79	13	4	105	0.9
	27			34			81	10	5
0.21	36	0.6	96	8	1.0	103	72	17	7	119	1.0
	32			30			79	12	5
0.62	35	0.6	93	11	1.0	102	73	16	7	137	1.1
	35			28			73	21	2
1.85	29	0.7	109	6	1.2	118	80	6	6	84	0.7
	30			24			78	12	6
2.78	24	0.8	124	7	1.0	100	81	8	7	68	0.6
	26			33			87	6	3
5.56	31	0.8	122	32	0.7	72	71	17	10	179	1.5
	20			30			78	7	11
MMS	44	0.6	86	34	0.5	49	49	16	31	658	5.4
25 µg/ml	32			55			53	21	23

0: vehicle control ( DMSO )

MMS:methylmethanesulfonate

LC: large colonies

CE₀and CE₂: cloning efficiency

SC: small colonies

RCE₀and RCE₂: relative cloning efficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

Table : First experiment without S9 mix, 3-hour treatment: cell growth during expression time

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	2.7	2.0	11.0	2.0	4.7	5.5	2.4	13.0
0									100	100
	3.6	2.0	7.5	2.0	6.3	3.8	3.2	11.8
	3.1	2.0	7.1	2.0	6.0	3.5	3.0	10.5
0.07									76	67
	3.7	2.0	6.2	2.0	5.4	3.1	2.7	8.4
	3.9	2.0	5.6	2.0	5.3	2.8	2.7	7.4
0.21									72	74
	3.5	2.0	7.1	2.0	5.9	3.6	2.9	10.4
	4.2	2.0	5.0	2.0	5.9	2.5	3.0	7.3
0.62									69	70
	3.9	2.0	6.9	2.0	5.7	3.4	2.9	9.8
	3.2	2.0	6.2	2.0	6.4	3.1	3.2	9.8
1.85									69	82
	3.1	2.0	5.3	2.0	5.6	2.6	2.8	7.3
	2.6	2.0	5.6	2.0	5.6	2.8	2.8	7.8
2.78									61	61
	2.3	2.0	5.3	2.0	5.6	2.7	2.8	7.4
	0.2	0.2 *	0.3	0.3 *	0.7	1.7	2.7	4.5
5.56									44	31
	0.1	0.1 *	0.1	0.1 *	0.4	2.0	3.1	6.3
	3.8	2.0	4.3	2.0	5.5	2.2	2.7	5.9
MMS									60	29
25 µg/ml	3.1	2.0	6.5	2.0	5.6	3.2	2.8	8.9

0: vehicle control ( DMSO )

conc.: cell concentration before being adjusted

adj.conc.:adjusted cell concentration when replating

SG: suspension growth

RSG: relative suspension growth

RTG: relative total growth

MMS: methylmethane sulfonate

*: cells were not replated as their density was < 2 x 10⁵/ml

Table: First experiment without S9 mix, 3-hour treatment: toxicity immediately after treatment

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	2.7
0		1.0	0.7	0.7	100
	3.6
	3.1
0.07		1.1	0.8	0.9	130
	3.7
	3.9
0.21		1.2	0.6	0.8	115
	3.5
	4.2
0.62		1.3	0.6	0.8	121
	3.9
	3.2
1.85		1.0	0.7	0.7	110
	3.1
	2.6
2.78		0.8	0.8	0.7	97
	2.3
	0.2
5.56		0.0	0.8	0.0	4
	0.1
	3.8
MMS		1.1	0.6	0.6	95
25 µg/ml	3.1

0: vehicle control (DMSO )

CE0: cloning efficiency immediately after treatment

RS: relative survival

MMS: methylmethane sulfonate

Table : Second experiment without S9 mix, 24-hour treatment: mutagenicity results

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF10-6	R
	35			34			85	6	5
0	34	0.7	100	36	0.7	100	84	8	4	124	1.0
	32			32			80	12	4
	28			32			77	11	8
0.06	28	0.6	95	28	0.8	124	82	10	5	85	0.7
	40			24			85	7	5
0.19	34	0.7	101	31	0.7	103	82	8	7	81	0.7
	30			34			90	6	0
0.56	36	0.6	91	31	0.7	103	84	7	5	64	0.5
	35			34			92	3	1
1.67	35	0.4	54	23	0.7	107	85	7	4	74	0.6
	71			39			88	1	7
2.5	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
5	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
MMS	29	0.8	122	36	0.6	97	56	16	24	394	3.2
5 µg/ml	22			33			60	23	15

0: vehicle control ( DMSO )

MMS: methylmethanesulfonate           

LC: large colonies

CE₀and CE₂: cloning efficiency

SC: small colonies

RCE₀and RCE₂: relative cloning efficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

-: not evaluated due to severe toxicity observed

Table : Second experiment without S9 mix, 24-hour treatment: cell growthduring expressiontime

 

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	5.8	2.0	6.8	2.0	6.3	3.4	3.1	10.5
0									100	100
	8.4	2.0	8.4	2.0	5.5	4.2	2.8	11.6
	6.2	2.0	5.5	2.0	5.7	2.8	2.9	7.8
0.06									84	104
	5.2	2.0	7.1	2.0	6.1	3.6	3.0	10.7
	5.5	2.0	6.2	2.0	5.0	3.1	2.5	7.7
0.19									79	82
	5.4	2.0	6.8	2.0	5.8	3.4	2.9	9.9
	4.1	2.0	7.4	2.0	6.0	3.7	3.0	10.9
0.56									101	104
	2.6	2.0	6.3	2.0	7.4	3.1	3.7	11.5
	0.0	0.0 *	0.2	0.2 *	0.4	4.8	2.1	9.8
1.67									59	63
	0.1	0.1 *	0.1	0.1 *	0.3	0.7	4.7	3.3
	-	-	-	-	-	-	-	-
2.5									-	-
	-	-	-	-	-	-	-	-
	-	-	-	-	-	-	-	-
5									-	-
	-	-	-	-	-	-	-	-
	7.8	2.0	7.7	2.0	4.8	3.9	2.4	9.2
MMS									82	80
5 µg/ml	7.4	2.0	7.0	2.0	5.1	3.5	2.6	8.9

0:vehiclecontrol(DMSO)

conc.: cell concentration before being adjusted

adj.conc.:adjusted cell concentration when replating

SG: suspension growth

RSG: relative suspension growth

RTG: relative total growth

MMS: methylmethane sulfonate

*: cells were not replated as their density was < 2 x 10⁵/ml

-: not evaluated due to severe toxicity observed

Table : Second experiment without S9 mix, 24-hour treatment: toxicity immediately after treatment

 

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	5.8
0		1.0	0.7	0.7	100
	8.4
	6.2
0.06		0.8	0.6	0.5	76
	5.2
	5.5
0.19		0.8	0.7	0.5	77
	5.4
	4.1
0.56		0.5	0.6	0.3	43
	2.6
	0.0
1.67		0.0	0.4	0.0	0
	0.1
	-
2.5		-	-	-	-
	-
	-
5		-	-	-	-
	-
	7.8
MMS		1.1	0.8	0.9	130
5 µg/ml	7.4

0: vehicle control ( DMSO )

CE0: cloning efficiency immediately after treatment

RS: relative survival

MMS: methylmethane sulfonate

-: not evaluated due to severe toxicity observed

Table: First experiment with S9 mix, 3-hour treatment: mutagenicity results

 

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF10-6	R
	8			10			77	11	8
0	6	1.4	100	14	1.3	100	82	9	5	75	1.0
	17			21			80	11	5
	12			4			78	9	9
0.21	5	2.1	151	5	1.8	139	83	9	4	46	0.6
	2			6			80	11	5
0.62	5	1.7	123	11	1.4	112	77	15	4	52	0.7
	8			8			88	6	2
1.85	3	2.1	151	4	1.6	127	76	9	11	56	0.7
	4			10			84	2	10
5.56	13	1.4	106	16	1.0	80	85	2	9	45	0.6
	6			21			90	2	4
16.7	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
50	-	-	-	-	-	-	-	-	-	-	-
	-			-			-	-	-
CPA	13	1.1	78	31	0.7	56	41	27	29	491	6.6
3 µg/ml	22			30			54	13	30

0: vehicle control ( DMSO )

CPA:cyclophosphamide          

LC: large colonies

CE₀and CE₂:cloningefficiency

SC: small colonies

RCE₀and RCE₂: relative cloningefficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

-: not evaluated due to severe toxicity observed

Table : First experiment with S9 mix, 3-hour treatment: cell growth during expression time

 

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	3.8	2.0	8.0	2.0	7.3	4.0	3.6	14.5
0									100	100
	4.4	2.0	6.9	2.0	7.2	3.5	3.6	12.4
	4.2	2.0	7.2	2.0	7.5	3.6	3.7	13.4
0.21									113	157
	4.1	2.0	8.5	2.0	8.1	4.2	4.0	17.0
	4.3	2.0	9.1	2.0	5.4	4.5	2.7	12.2
0.62									103	116
	3.6	2.0	9.2	2.0	6.8	4.6	3.4	15.5
	3.8	2.0	6.1	2.0	5.6	3.0	2.8	8.5
1.85									67	85
	4.8	2.0	5.1	2.0	7.5	2.5	3.8	9.5
	3.1	2.0	6.5	2.0	3.1	3.2	1.5	5.0
5.56									38	31
	3.4	2.0	4.3	2.0	5.0	2.2	2.5	5.4
	0.0	-	-	-	-	-	-	-
16.7									-	-
	0.0	-	-	-	-	-	-	-
	0.0	-	-	-	-	-	-	-
50									-	-
	0.0	-	-	-	-	-	-	-
	3.8	2.0	6.1	2.0	6.1	3.1	3.0	9.2
CPA									82	46
3 µg/ml	3.9	2.0	5.6	2.0	9.3	2.8	4.6	13.0

0: vehicle control ( DMSO )

conc.: cell concentration before being adjusted

adj.conc.:adjustedcellconcentrationwhenreplating SG: suspensiongrowth

RSG: relative suspension growth RTG: relative total growth

CPA: cyclophosphamide

-: not evaluated due to severe toxicity observed

Table : First experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment

 

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	3.8
0		1.0	1.4	1.4	100
	4.4
	4.2
0.21		1.0	2.1	2.1	153
	4.1
	4.3
0.62		1.0	1.7	1.6	119
	3.6
	3.8
1.85		1.0	2.1	2.2	158
	4.8
	3.1
5.56		0.8	1.4	1.1	83
	3.4
	0.0
16.7		0.0	-	-	-
	0.0
	0.0
50		0.0	-	-	-
	0.0
	3.8
CPA		0.9	1.1	1.0	73
3 µg/ml	3.9

0: vehicle control ( DMSO )

CE0: cloning efficiency immediately after treatment RS: relative survival

CPA: cyclophosphamide

-: not evaluated due to severe toxicity observed

Table : Second experiment with S9 mix, 3-hour treatment: mutagenicity results

 

Doses µg/ml (a.i.)	Empty wells*	CE0	RCE0 %	Empty wells*	CE2	RCE2 %	Empty wells*	LC	SC	MF 10-6	R
	24			12			82	9	5
0	30	0.8	100	21	1.1	100	82	9	5	67	1.0
	23			7			82	9	5
	30			25			85	5	6
0.19	30	0.8	96	10	1.4	122	80	9	9	60	0.9
	26			12			83	10	3
0.56	28	0.8	101	6	1.3	117	82	12	2	61	0.9
	25			18			82	9	5
1.67	17	0.9	110	6	1.4	127	83	9	4	67	1.0
	30			14			76	14	6
5	32	0.7	92	3	2.0	179	79	9	8	57	0.9
	27			5			74	14	9
	40			5			80	11	5
7.5		0.6	74		1.6	144				48	0.7
	35			10			85	6	5
10	49	0.4	53	26	0.7	64	91	5	0	26	0.4
	48			36			94	2	0
CPA	38	0.5	65	26	0.7	63	60	15	22	440	6.6
3 µg/ml	46			37			44	19	37

0: vehicle control ( DMSO )

CPA:cyclophosphamide          

LC: largecolonies

CE₀and CE₂:cloning efficiency

SC: small colonies

RCE₀and RCE₂: relative cloningefficiency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

*: empty wells are counted on a total number of 96 wells

Table : Second experiment with S9 mix, 3-hour treatment: cell growth during expression time

 

Doses µg/ml (a.i.)	Cell concentration (x 10^+5/ml)					Daily cell growth			RSG %	RTG %
	Day 0		Day 1		Day 2	Day 1	Day 2	Day 1x Day 2 (SG)
	Conc.	Adj. Conc.	Conc.	Adj. Conc.	Conc.
	5.7	2.0	6.9	2.0	9.6	3.5	4.8	16.5
0									100	100
	5.6	2.0	7.0	2.0	7.9	3.5	4.0	13.7
	5.2	2.0	8.3	2.0	6.3	4.1	3.2	13.0
0.19									84	102
	5.1	2.0	7.5	2.0	6.6	3.8	3.3	12.4
	5.7	2.0	8.0	2.0	5.9	4.0	3.0	11.7
0.56									74	87
	6.5	2.0	5.4	2.0	8.0	2.7	4.0	10.7
	4.9	2.0	7.5	2.0	5.1	3.7	2.5	9.4
1.67									57	73
	6.5	2.0	5.3	2.0	6.0	2.6	3.0	7.8
	6.6	2.0	3.6	2.0	5.1	1.8	2.6	4.6
5									29	52
	5.5	2.0	5.2	2.0	3.3	2.6	1.6	4.2
	4.6	2.0	2.0	2.0 *	2.0	1.0	1.0	1.0
7.5									9	13
	5.1	2.0	2.4	2.0	2.7	1.2	1.3	1.6
	2.7	2.0	2.0	2.0 *	1.1	1.0	0.5	0.6
10									4	2
	2.4	2.0	1.4	1.4 *	1.1	0.7	0.8	0.5
	6.4	2.0	5.5	2.0	7.1	2.8	3.5	9.7
CPA									61	38
3 µg/ml	6.7	2.0	6.2	2.0	5.7	3.1	2.9	8.8

0: vehicle control ( DMSO )

conc.: cell concentration before being adjusted

adj.conc.:adjusted cell concentration when replating

SG: suspensiongrowth

RSG: relative suspension growth

RTG: relative total growth

CPA: cyclophosphamide

*: cells were not replated as their density was < 2 x 10⁵/ml

Table : Second experiment with S9 mix, 3-hour treatment: toxicity immediately after treatment

  

Doses µg/ml (a.i.)	post-treatment cell count (x 10^5cells/ml)	Cell count factor	CE0	Survival	RS %
	5.7
0		1.0	0.8	0.8	100
	5.6
	5.2
0.19		0.9	0.8	0.7	87
	5.1
	5.7
0.56		1.1	0.8	0.9	109
	6.5
	4.9
1.67		1.0	0.9	0.9	111
	6.5
	6.6
5		1.1	0.7	0.8	98
	5.5
	4.6
7.5		0.9	0.6	0.5	63
	5.1
	2.7
10		0.4	0.4	0.2	24
	2.4
	6.4
CPA		1.2	0.5	0.6	75
3 µg/ml	6.7

0: vehicle control ( DMSO )

CE0: cloning efficiency immediately after treatment

RS: relative survival

CPA: cyclophosphamide

Table: Historical data without S9 mix - Mouse lymphoma assay

	3-hour treatment						24-hour treatment
assay No.	vehicle			MMS			vehicle			MMS
	CE0	CE2	MF	CE0	CE2	MF	CE0	CE2	MF	CE0	CE2	MF
1	1.2	0.9	84	0.8	0.5	682	0.6	0.8	118	0.4	0.6	539
2	0.9	0.7	82	0.9	0.5	528	0.6	0.8	128	0.5	0.5	603
3	1.1	0.9	139	1	0.6	467	0.7	0.9	242	0.6	0.6	601
4	-	-	-	-	-	-	0.7	0.7	78	0.5	0.5	475
5	0.7	0.8	124	0.8	0.4	499	0.7	0.7	126	0.5	0.6	479
6	0.9	0.7	117	0.7	0.5	424	0.6	0.8	143	0.4	0.5	755
7	0.9	0.8	108	0.7	0.5	431	0.7	0.8	72	1.1	0.6	426
8	0.6	0.7	126	0.6	0.4	612	0.6	0.7	161	0.4	0.5	990
9	1.0	0.8	130	1.1	0.5	605	-	-	-	-	-	-
10	1.1	1.1	61	1.1	0.5	861	0.7	1.1	87	1.1	0.7	675
11	1.2	0.9	75	0.9	0.5	458	0.6	0.7	125	0.5	0.4	1298
12	1.3	0.9	58	1.8	0.7	462	1.0	1.3	94	0.8	0.5	823
13	1.0	1.3	77	1.2	0.6	533	1.0	1.3	94	0.8	0.5	823
minimum	0.6	0.7	58	0.6	0.4	424	0.6	0.7	72	0.4	0.4	426
maximum	1.3	1.3	139	1.8	0.7	861	1.0	1.3	242	1.1