Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +/- S9, negative, Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2 (pKM101) and WP2 uvrA (pKM101), according to OECD TG 471, Callander 2006

- In vitro chromosome aberration study, +/- S9, negative, human peripheral blood lymphocytes, according to OECD TG 473, Fox 2006

- In vitro gene mutation assay, +/- S9, negative, mouse lymphoma L5178Y cells TK+/-, according to OECD TG 476, Clay 2006

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:

27 Jan 2006 to 27 Feb 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium) and trp- (E.coli) strains)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : phenobarbital/β-naphthoflavone induced rat liver
- Concentration or volume of S9 mix and S9 in the final culture medium: The S9 is prepared from male rats (Sprague-Dawley) induced with 80 mg/kg bw phenobarbital and 100 mg/kg bw β-naphthoflavone (3 mL of phenobarbital and β-naphthoflavone in total) combined with 7 mL Sucrose-Tris-EDTA buffer and 20 mL cofactor solution. The cofactor solution was prepared as a single stock solution of Na2HPO4 (150mM), KCl (49.5mM), glucose-6-phosphate (7.5mM), NADP (Na salt) (6mM) and MgCl2 (12mM) in sterile double deionised water and adjusted to a final pH of 7.4.
- Quality controls of S9: Positive control substances are tested to confirm the activity of the S9-mix.

Test concentrations with justification for top dose:

100, 200, 500, 1000, 2500, 5000 μg/plate

Vehicle / solvent:

- Solvent used: dried dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: Daunorubicin hydrochloride: 1.0 μg/plate in TA98 (without metabolic activation), 2-Aminoanthracene: 1.0 μg/plate in TA100 and TA98; 2.0 μg/plate in TA1535 and TA1537; and 20 μg/plate in WP2 (pKM101) (with metabolic activation)

Details on test system and experimental conditions:

DOSING PREPARATIONS
All test and positive control substance dosing preparations were prepared as close to the time of culture treatment as possible and are dosed at a dosing volume of 100 μL/plate (apart from in the pre-incubation experiment, where the volume is reduced to 20 μL/plate).

EXPERIMENTAL PERFORMANCE
Bacterial cultures were prepared from frozen stocks by incubating for 10-12 hours at 37°C in a shaking incubator. The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level, solvent and positive controls;
- 500 μL S9 mix or phosphate buffer;
- 100 μL Bacteria suspension;
- 2 mL Overlay agar containing 50 μM histidine or tryptophan as appropriate.
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix and 100 μL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After the agar was set the plates were incubated upside down for 3 days at 37° C in the dark.
For each strain and dose level, including the controls three plates were used.
Following incubation all plates were counted using an automated colony counter adjusted appropriately to permit the optimal counting of mutant colonies.

CYTOTOXICITY
Following the total incubation period the plates were examined for the lack of microbial contamination and evidence that the test was valid: i.e. there was a background lawn on the solvent control plates and on the plates for (at least) the lower concentrations of test substance, and that the positive controls had responded as expected. Except where prohibited by the density of the precipitate the number of revertant colonies on each plate was counted using an automated adjusted appropriately to permit the optimal counting of mutant colonies. Plates that were obviously contaminated were recorded as such without being scored.

PRE-INCUBATION PROTOCOL
The assay procedure was as for the plate-incorporation protocol described above, except that a) each compound/ solvent dose was added in 0.02 mL volumes, with the total volume made up to 0.1 mL with phosphate buffered saline; b) before adding the top agar, each compound/strain group of containers were placed on an orbital shaker for 60 minutes (at 37°C).

DATA PRESENTATION
The data reported included individual numbers of revertants per plate, mean values and standard deviation for each bacterial strain and each concentration. Historical values of negative controls obtained with each strain without and with microsomal activation were listed in a separate table, which contains mean values of controls as well as standard deviations and acceptable minimal and maximal values of spontaneous revertants for each bacterial strain.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
a) a significant, dose-related increase in the mean number of revertants is observed;
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations

CRITERIA FOR A NEGATIVE RESPONSE
A negative result in a (valid) individual experiment is achieved when:
a) there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

CRITERIA FOR VALID TEST DATA
Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable;
b) the positive control data show acceptable increases;

Key result

Species / strain:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
Precipitation occurred at concentrations of 500-5000 μL/0.1mL. Incomplete background lawns were reported only at a concentration of 200 μL/0.1mL on the E. coli WP2 (pKM101) plates.

MUTAGENICITY
In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix.
The positive controls for each experiment induced the expected responses indicating the strains were responding satisfactorily in each case.

Conclusions:

The test material gave a negative (non-mutagenic) response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strains WP2 (pKM101) and WP2 uvrA (pKM101) in both the presence and absence of S9-mix.

Executive summary:

The test material was evaluated in a bacterial reverse mutation assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2 (pKM101) and WP2 uvrA (pKM101) in the presence and absence of a rat liver-derived metabolic activation system (S9-mix). The investigations were performed with concentrations of 100, 200, 500, 1000, 2500, 5000 μg/plate in all experiments. This study was conducted in accordance with OECD TG 471 following GLP principles.

In two independent experiments, the test material did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the strains used, either in the presence or absence of S9-mix. The sensitivity of the test system, and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

The test material gave a negative (non-mutagenic) response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strains WP2 (pKM101) and WP2 uvrA (pKM101) in both the presence and absence of S9-mix.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan 2006 to 15 Mar 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

primary culture, other: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Human blood samples were obtained by venepuncture in lithium heparin tubes on the days of culture initiation from healthy, non-smoking donors. Equal volumes of blood from 2 male donors were pooled together for Experiment 1 and equal volumes of blood from 2 female donors were pooled together for Experiment 2. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.
At 0 hours, cultures (10 mL) were established by the addition of 0.5 mL of whole blood to RPMI-1640 tissue culture medium supplemented with approximately 10 %
foetal bovine serum (FBS), 1.0 IU/mL heparin, L-glutamine (2 mM), 100 IU/mL penicillin and 100 μg/mL streptomycin. The lymphocytes were stimulated to enter cell division by addition of phytohaemagglutinin (PHA; at 5 % v/v) and the cultures were maintained at approximately 37°C for 48 hours with gentle daily mixing.

Cytokinesis block (if used):

Two hours before harvesting, the cultures were treated with Colcemide (0.4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Phenobarbital and β-naphthoflavone induced male Sprague Dawley rats
- Concentration or volume of S9 mix and S9 in the final culture medium: The S9 is prepared from male Sprague Dawley rats, dosed once daily (by oral gavage) for 3 days with a combined phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw). For the experiment, 200 μL of a 1:1 mix of S9 and co-factor solution is used.

Test concentrations with justification for top dose:

Experiment 1:
+S9-mix: 20, 30, 50 μg/ mL;
-S9-mix: 20, 30, 40 μg/ mL
Experiment 2:
+S9-mix: 20, 30, 50 μg/ mL;
-S9-mix: 10, 15, 20 μg/ mL

Vehicle / solvent:

- Solvent used: dried dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DOSING PREPARATIONS
An individual stock solution of the test material was prepared for each experiment in DMSO and serial dilutions were carried out as required in each case. Both positive control substances were prepared as solutions in sterile double deionised water. All test and positive control substance dosing preparations were prepared as close to the time of culture treatment as possible and were dosed at 100 μL/10mL culture.

EXPERIMENTAL DESIGN
Duplicate human peripheral blood cultures were exposed to the solvent, test material or positive control substances at appropriate concentrations in the following experiments:
a) A cytogenetic experiment was conducted using a sample of pooled blood. Cells were exposed to the test material and control substances for a period of 3 hours, both in the presence and absence of S9-mix. Solvent, untreated and positive control cultures were included.
b) A second independent cytogenetic experiment was conducted using a sample of pooled blood. Cells were exposed to the test material and control substances for a period of 3 hours in the presence of S9-mix and 20 hours in the absence of S9-mix. Solvent, untreated and positive control cultures were included. Treatment of the cultures started approximately 48 hours after culture initiation. A single sampling time, 20 hours after the start of treatment was used. The sampling time of 20 hours after the start of treatment, used in this study, was based on a measured mean cell cycle time for cultured human peripheral blood lymphocytes of 13.5 hours in this Laboratory.

CULTURE TREATMENT
Prior to treatment, the cultures to be treated for a 20 hour period were centrifuged and the culture medium was replaced with fresh supplemented RPMI-1640 culture medium. The cultures to be treated for a 3 hour period did not receive a medium change.
Approximately 48 hours after culture establishment, aliquots of the test material, solvent control or positive controls were administered to duplicate cultures. In addition, 200 μL of a 1:1 mix of S9 and co-factor solution was added to each culture to be treated in the presence of S9-mix. Cultures from both experiments in the presence of S9-mix and Experiment 1 in the absence of S9-mix, are treated for a period of 3 hours at 37°C, after which the culture medium was removed following centrifugation and replaced with fresh supplemented RPMI-1640 culture medium. The cultures were re-incubated at 37°C for the remainder of the 68 hour growth period. Cultures from Experiment 2 in the absence of S9-mix were treated for a period of 20 hours until the end of the 68 hour growth period.
The effects on the pH and osmolality of the culture medium was investigated, using single cultures containing medium only. The solubility of the test material in the treated blood cultures and in media only cultures was assessed by eye immediately after treatment and at the end of the treatment.

CULTURE HARVESTING
Approximately 2 hours prior to harvesting, the cultures were treated with colcemid at a final concentration of 0.4 μg/mL. Sixty-eight hours after culture establishment, the cultures were centrifuged, the supernatant removed and the cells were re-suspended in 10 mL of 0.075 M KCl for 10 minutes. The cultures were centrifuged, the supernatant was removed and the remaining cells were fixed in freshly prepared methanol/ glacial acetic acid fixative. The fixative was removed following centrifugation and replaced with freshly prepared fixative. After at least two subsequent changes of fixative, slides were made by dropping the cell suspension on to clean, moist labelled microscope slides. The slides were air dried, stained in filtered Giemsa stain for 7 minutes, rinsed in water, air-dried and mounted with coverslips in DPX.

SLIDE ANALYSIS
Slides were examined to determine that they were of suitable quality and, where appropriate, the mitotic index was determined by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase.
For each experiment, both in the presence and absence of S9-mix, duplicate cultures treated with the test material at 3 concentrations were selected for chromosomal aberration analysis along with the solvent and positive control cultures.
The slides were coded prior to analysis and one hundred cells in metaphase, where possible, were analysed from each selected culture for the incidence of structural chromosomal damage.

Evaluation criteria:

Evaluation of Clastogenicity:
a) No statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values indicates the study to be negative (non-clastogenic).
b) A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range indicates the study to be negative (non-clastogenic).
c) An increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values indicates the study to be positive (clastogenic).
d) A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described in (c) may require further evaluation.

Statistics:

The Fisher Exact Probability Test (one-sided) was used to evaluate statistically the percentage of metaphases showing aberrations (excluding cells with only gap-type aberrations). Data from each treatment group, in the presence and absence of S9-mix, was compared with the respective solvent control group value.

Key result

Species / strain:

primary culture, other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
The test material was found to be biologically active in the test system, causing concentration related reductions in mitotic activity.

CLASTOGENIC
In Experiment 1, there were small but statistically significant increases in the percentage of aberrant cells in the absence of S9-mix at 20 μg/mL and 30 μg/mL. There were no increases in the percentage of aberrant cells at 40 μg/mL and the increases at 20 μg/mL and 30 μg/mL were within the historical control solvent control range for this laboratory.
No other statistically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded in the study. The positive control materials, mitomycin C and cyclophosphamide induced statistically and biologically significant increases in the percentage of aberrant cells, compared to the solvent control cultures.

CYTOTOXICITY

Two independent cytogenetic experiments were carried out using ranges of the test materials concentrations. The highest concentrations selected for chromosome aberration analysis were based on reductions in mitotic activity.

Significant reductions in mean mitotic activity, compared to the solvent control values, were observed in cultures from both Experiment 1 (58 % +S9-mix; 52 % -S9-mix) and Experiment 2 (61 % +S9-mix; 55 % -S9-mix) treated with the highest concentrations of the test material selected for chromosomal aberration analysis. Cultures treated with higher concentrations of the test material were considered not to be suitable for chromosomal aberration analysis due to lack of metaphases as a result of toxicity.

Treatment of the culture medium with the test material up to 90 μg/mL had no significant effect on osmolality or pH.

Table 2. MITOTIC INDICES IN THE ABSENCE OF METABOLIC ACTIVATION (S9-MIX).

Experiment 1			Experiment 2
Treatment	Mitotic Index %	Mean % Mitotic Index	Treatment	Mitotic Index %	Mean % Mitotic Index
Solvent Control (10 μL/mL)	7.5 8.8		Solvent Control (10 μL/mL)	6.4 6.0
Test material (90 μg/mL)	A A		Test material (50 μg/mL)	A A
Test material (80 μg/mL)	A A		Test material (40 μg/mL)	A A
Test material (70 μg/mL)	A A		Test material (30 μg/mL)	A A
Test material (60 μg/mL)	A A		Test material (25 μg/mL)	A A
Test material (50 μg/mL)	2.4 2.9	2.7	Test material (20 μg/mL)	2.2 3.3	2.8
Test material (40 μg/mL)	4.7 3.0	3.9	Test material (15 μg/mL)	2.9 3.8	3.4
Test material (30 μg/mL)	4.5 3.9	4.2	Test material (10 μg/mL)	5.0 4.6	4.8
Test material (20 μg/mL)	6.1 5.4	5.8	Test material (5 μg/mL)	5.2 4.2	4.7

a - None of too few metaphases for chromosomal aberration analysis.

Table 3. MITOTIC INDICES IN THE PRESENCE OF METABOLIC ACTIVATION (S9-MIX).

Experiment 1			Experiment 2
Treatment	Mitotic Index %	Mean % Mitotic Index	Treatment	Mitotic Index %	Mean % Mitotic Index
Solvent Control (10 μL/mL)	7.2 8.9 7.9 7.2	7.8	Solvent Control (10 μL/mL)	8.0 4.4	6.2
Test material (90 μg/mL)	A A		Test material (80 μg/mL)	A A
Test material (80 μg/mL)	A A		Test material (70 μg/mL)	A A
Test material (70 μg/mL)	A A		Test material (60 μg/mL)	A A
Test material (60 μg/mL)	1.8 1.8	1.8	Test material (50 μg/mL)	2.9 1.8	2.4
Test material (50 μg/mL)	3.1 3.5	3.3	Test material (40 μg/mL)	3.4 4.7	4.1
Test material (40 μg/mL)	5.4 3.6	4.5	Test material (30 μg/mL)	3.0 4.7	3.9
Test material (30 μg/mL)	5.8 4.3	5.1	Test material (20 μg/mL)	7.5 7.1	7.3
Test material (20 μg/mL)	7.0 6.2	6.6	Test material (10 μg/mL)	B B

a - None or too few metaphases of chromosomal aberration analysis

b - Mitotic index not required for selection of concentrations for chromosome aberration analysis

Table 4. MEAN CHROMOSOMAL ABERRATIONS AND MITOTIC INDICES IN THE ABSENCE OF METABOLIC ACTIVATION (S9-MIX).

	Treatment	Mean % Aberrant Cells Excluding Gaps	Mean % Mitotic Index
Experiment 1– 3 hour treatment	Solvent Control (10 μg/mL)	0	8.2
	Mitomycin C (0.5 μg/mL)	40.00**	2.4Δ
	Test material (40 μg/mL)	1.50	3.9
	Test material (30 μg/mL)	3.00*	4.2
	Test material (20 μg/mL)	2.50*	5.8
Experiment 2– 20 hour treatment	Solvent Control (10 μg/mL)	1.50	6.2
	Mitomycin C (0.2 μg/mL)	36.00**	4.8Δ
	Test material (20 μg/mL)	1.50	2.8
	Test material (15 μg/mL)	4.00	3.4
	Test material (10 μg/mL)	1.50	4.8

* Statistically significant increase in the percentage of aberrant cells at p<0.05 using Fisher's Exact Test (one-sided).

** Statistically significant increase in the percentage of aberrant cells at p<0.01 using Fisher's Exact Test (one-sided).

Δ Positive control mitotic index and % aberrant cells are determined from a single culture.

Table 5. MEAN CHROMOSOMAL ABERRATIONS AND MITOTIC INDICES IN THE PRESENCE OF METABOLIC ACTIVATION (S9-MIX).

	Treatment	Mean % Aberrant Cells Excluding Gaps	Mean % Mitotic Index
Experiment 1– 3 hour treatment	Solvent Control (10 μg/mL)	3.06	7.8
	Cyclophosphamide (50 μg/mL)	48.00**	2.4Δ
	Test material (50 μg/mL)	1.00	3.3
	Test material (30 μg/mL)	1.50	5.1
	Test material (20 μg/mL)	0.50	6.6
Experiment 2– 20 hour treatment	Solvent Control (10 μg/mL)	4.50	6.2
	Cyclophosphamide (50 μg/mL)	40.00**	2.4Δ
	Test material (50 μg/mL)	5.50	2.4
	Test material (30 μg/mL)	5.00	3.9
	Test material (20 μg/mL)	3.50	7.3

** Statistically significant increase in the percentage of aberrant cells at p<0.01 using Fisher's Exact Test (one-sided).

Δ Positive control mitotic index and % aberrant cells are determined from a single culture.

Conclusions:

The test material is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.

Executive summary:

The test material was evaluated for its clastogenic potential in an in vitro cytogenetic assay using human lymphocytes in two independent experiments treated in the presence and absence of a rat liver-derived metabolic activation system (S9-mix). This study was conducted in accordance with OECD TG 473 following GLP guidelines. In Experiment 1 cultures were treated for a period of 3 hours both in the presence and absence of S9-mix. In Experiment 2 cultures were treated for a period of 3 hours in the presence of S9-mix and 20 hours in the absence of S9-mix. All cultures were harvested 68 hours after culture initiation. The concentrations were chosen to be 20, 30, 50 μg/ mL (+S9 -mix) and 20, 30, 40 μg/ mL (-S9 -mix) in Experiment 1, and 20, 30, 50 μg/ mL (+S9 -mix) and 10, 15, 20 μg/ mL (-S9 -mix) in Experiment 2. Cultures were treated with the test material at appropriate concentrations for chromosomal aberration analysis along with the appropriate solvent and positive control cultures.

The highest concentrations selected for chromosome aberration analysis were based on reductions in mitotic activity. Concentration related reductions in mitotic activity were observed in cultures from both experiments, thus demonstrating that the test material is biologically active in this test system. In Experiment 1, there were small but statistically significant increases in the percentage of aberrant cells in the absence of S9-mix at 20 μg/mL and 30 μg/mL There were no increases in the percentage of aberrant cells at 40 μg/mL and the increases at 20 μg/mL and 30 μg/mL were within the historical control solvent control range for this laboratory. No other statistically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded. The sensitivity of the test system, and the metabolic activity of the S9-mix, were demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents.

The test material is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan 2006 to 13 Mar 2006

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)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

L5178Y TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

A bank of L5178Y TK+/- 3.7.2.c cells was stored in a liquid nitrogen freezer. The cell stocks have been shown to be free of mycoplasma. Following removal from liquid nitrogen, the cultures were kept at 37 °C under an atmosphere of 5 % CO2 in air in a gassing incubator, or in a hot room in roller bottles rotated on a roller apparatus.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital and β-naphthoflavone induced male Sprague Dawley rats
- Concentration or volume of S9 mix and S9 in the final culture medium: The S9 is prepared from male Sprague Dawley rats, dosed once daily (by oral gavage) for 3 days with a combined phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw). The co-factor solution and S9 fraction are added at 1% (200 µL of each added to the 20 mL cell culture).

Test concentrations with justification for top dose:

Experiment 1:
+S9-mix: 2.5, 5, 10, 20, 30, 40, 50 μg/ mL;
-S9-mix: 0.63, 1.25, 2.5, 5, 10, 20, 30 μg/ mL
Experiment 2:
+S9-mix: 5, 10, 15, 20, 25, 30 μg/ mL;
-S9-mix: 1, 2, 5, 10, 15, 20 μg/ mL
Experiment 3:
+S9-mix: 15, 20, 25, 30, 35, 40 μg/ mL;
-S9-mix: 2, 5, 10, 15, 20, 25 μg/ mL

Vehicle / solvent:

- Solvent used: dimethylsulphoxide (DMSO) 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

DOSING PREPARATIONS
An individual stock of the test material was prepared for each experiment in DMSO and dilutions were carried out. All test and positive control substance dosing preparations were prepared as close to the time of culture treatment as possible and were dosed at 10 μL/mL culture.

EXPERIMENTAL DESIGN
The test material was tested both in the presence and absence of S9-mix in three independent experiments.Two series of exponentially growing suspension cultures of L5178Y cells were treated in duplicate with the solvent control, positive controls or a range of concentrations of the test material for 4 hours in the presence and absence of S9-mix. After removal of the treatment medium, the cells were cultured to allow any induced mutants to be expressed. The growth rate was monitored and the cells subcultured daily. After 48 hour expression time, samples were grown in both selective and non selective medium, and the results obtained used to determine the mutant frequency per viable cell.

CELL PREPARATION
A fresh sample of cells was brought up from liquid nitrogen storage for each experiment. A minimum of 10^7 cells in exponential growth were required per treatment, therefore, a bulk culture was prepared prior to each experiment and diluted with serum free medium to obtain a reduced serum content of 5 % at treatment time. Each 20 mL treatment culture was taken from this culture.

CULTURE TREATMENT
Cells were exposed to test compound, negative/solvent or positive controls for 4 hours in both the presence and absence of S9 mix. During this period the treated cell cultures were rotated on a roller apparatus in a 37°C hot room. Experiments were done in duplicates.
The effect on the pH and osmolality of the treatment medium was investigated.

SURVIVAL
Survival was measured by relative total growth (RTG). RTG is a measure of growth of test cultures both during the two-day expression and cloning phases of the assay, relative to the vehicle control.

EXPRESSION TIME
The post-treatment cultures were returned to the roller apparatus in the 37 °C hot room for a 48 hour expression period. To maintain exponential growth during the expression time, each culture was counted and, where appropriate, diluted daily to give approximately 2 x 10^5 cells per mL in 50 mL, thereby ensuring approximately 10^7 cells at each subculture.

MUTATION ASSAY
After the 48 hour expression period, the mutation assay was performed. The cell density of each culture was determined and the cultures were then divided into two series of dilutions, one for the assessment of mutants by TFT selection; the second to assess the viability of the cultures (absence of TFT). For the assessment of mutants, a sample of each of the post-expression cultures was diluted to give 50 mL at 1 x 10^4 cells per mL. TFT was then added to the mutation cultures. Each TFT treated culture was then dispensed at 200 μL per well into 2 x 96 well microwell plates (2000 cells per well). These plates were then incubated to allow cell growth. For the assessment of viability, a sample from each mutation culture (at 1 x 10^4/mL) was diluted to give 50 mL at 8 cells per mL. No TFT was added to these cultures. Each viability culture was then dispensed and incubated as for the mutation cultures.

EVALUATION OF RESULTS
Cell growth in individual microwell plates was assessed after 10 to 13 days using a dissecting microscope. The survival plates and viability plates were scored for the number of wells containing no cell growth (negative wells). The mutation plates were scored so that each well contained either a small colony, a large colony, or no colony.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
A significant dose-related increase in mutant frequency is required. Such an increase should be observed across a range of levels of toxicity and not exclusively at those concentrations eliciting high levels of toxicity. An associated absolute increase in mutant number above the solvent control values is a further requirement. Such a response must be reproducible in an independent experiment for the test material to be described as positive in this assay.

CRITERIA FOR SCORING MUTATION PLATES
- Small Colony : average diameter is < 25% of the diameter of the well and Is around 15% of the diameter of the well. A small colony should also have shown a dense clonal morphology.
- Large Colony: average diameter is > 25% of the diameter of the well. A large colony should also have shown less densely packed cells, especially around the edges of the colony. Any well which contained >1 small colony was scored as a small colony. Any well which contained >1 large colony was scored as a large colony. Any well which contained a combination of large and small colonies was scored as a large colony. An empty well was one which contained no cell growth.

CRITERIA FOR VALID TEST DATA
- Spontaneous control data: demonstrate acceptable cell growth and maintenance throughout the course of an experiment, adequate post-expression cloning efficiencies should be achieved for the solvent control viability plates. The spontaneous mutant frequency should be appropriate to the test conditions and be stable.
- Positive control data: relevant positive controls are used in the absence and presence of S9-mix i.e., ethylmethanesulphonate and benzo[a]pyrene respectively. These must give appropriate positive responses.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/-

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
Dose related toxicity was observed in all experiments. In experiment 1, the maximum concentrations evaluated for mutant frequency were 30 μg/mL and 10 μg/mL, resulting in 13% and 49% survival, relative to the control values in the presence and absence of S9-mix, respectively. In experiment 2 the maximum concentrations evaluated for mutant frequency were 30 μg/mL and 20 μg/mL, resulting in 28% and 18% survival in the presence and absence of S9-mix, respectively. In experiment 3 the maximum concentrations evaluated for mutant frequency were 40 μg/mL and 25 μg/mL, resulting in 14% and 19% survival in the presence and absence of S9-mix, respectively.

MUTAGENICITY
No significant increases in mutant frequency, compared to the solvent control cultures, were observed in cultures treated with the test material at any concentration tested in either the presence or absence of S9-mix. The positive controls, EMS and BP, induced appropriate increases in mutant frequency in all mutation experiments, demonstrating the activity of the S9-mix and that the assay was performing satisfactorily in being capable of detecting known mutagens. The data obtained in this study therefore show that the test material was not mutagenic in L5178Y TK+/- cells following in vitro treatment in either the presence or absence of S9-mix.

Table 1. Experiment 1 data.

Without S9-mix			With S9-mix
Concentration (μg/mL)	Mean % Relative Total Growth	Mean Mutant Frequency (x 10-4)	Concentration (μg/mL)	Mean % Relative Total Growth	Mean Mutant Frequency (x 10-4)
Test Material: 30	2	B	Test Material: 50	1	B
Test material: 20	6	B	Test material: 40	3	B
Test Material: 10	49	1.3	Test Material: 30	13	1.8
Test Material: 5	80	1.1	Test Material: 20	59	1.6
Test Material: 2.5	89	1.1	Test Material: 10	81	1.3
Test Material: 1.25	69	0.9	Test Material: 5	89	1.6
Test Material: 0.63	101	1.1	Test Material: 2.5	89	1.2
Solvent Control (DMSO): 10	100	1.0	Solvent Control (DMSO): 10	100	1.0
Positive Control (EMS): 500	41	6.5	Positive Control (BP): 1	75	4.3

b = not counted due to excessive toxicity

Table 2. Experiment 2 data.

Without S9-mix			With S9-mix
Concentration (μg/mL)	Mean % Relative Total Growth	Mean Mutant Frequency (x 10-4)	Concentration (μg/mL)	Mean % Relative Total Growth	Mean Mutant Frequency (x 10-4)
Test Material: 20	18	1.6	Test Material: 30	28	2.3
Test material: 15	49	1.1	Test material: 25	85	1.4
Test Material: 10	70	1.0	Test Material: 20	101	1.1
Test Material: 5	87	1.0	Test Material: 15	108	1,7
Test Material: 2	66	0.8	Test Material: 10	120	1.7
Test Material: 1	67	1.5	Test Material: 5	119	1.6
Solvent Control (DMSO): 10	100	0.9	Solvent Control (DMSO): 10	100	1.8
Positive Control (EMS): 500	33	9.9	Positive Control (BP): 1	72	8.4

Table 3. Experiment 3 data.

Without S9-mix			With S9-mix
Concentration (μg/mL)	Mean % Relative Total Growth	Mean Mutant Frequency (x 10-4)	Concentration (μg/mL)	Mean % Relative Total Growth	Mean Mutant Frequency (x 10-4)
Test Material: 25	19	1.5	Test Material: 40	14	1.5
Test material: 20	22	1.3	Test material: 35	28	0.9
Test Material: 15	50	0.8	Test Material: 30	42	1.3
Test Material: 10	81	0.9	Test Material: 25	51	0.7
Test Material: 5	94	1.1	Test Material: 20	81	0.6
Test Material: 2	94	0.7	Test Material: 15	78	1.1
Solvent Control (DMSO): 10	100	0.9	Solvent Control (DMSO): 10	100	1.0
Positive Control (EMS): 500	68	4.1	Positive Control (BP): 1	69	5.1

Conclusions:

The test material is not mutagenic in L5178Y TK+/- cells treated in vitro in either the presence or absence of S9-mix.

Executive summary:

To assess the potential of the test material to cause gene mutation or clastogenic effects in mammalian cells, L5178Y TK+/- mouse lymphoma cells were treated in vitro with various concentrations of the test substance, both in the presence and absence of a rat liver derived auxiliary metabolic system (S9-mix). This study was conducted in accordance with OECD TG 476 following GLP principles. Large and small mutant colonies were scored for all cultures in each experiment. Mutant frequencies were assessed by cell growth in the presence of trifluorothymidine after a 48 hour expression time. The test material was tested up to maximum concentrations of 40 μg/mL and 25 μg/mL in the presence and absence of S9-mix, respectively, in 3 independent experiments.

Minimum survival levels, compared to the solvent control cultures, of 14 % and 19 % were observed in cultures treated with the maximum concentrations of the test material in the presence and absence of S9-mix, respectively. No significant increases in mutant frequency were observed in cultures treated with the test material in either the presence or absence of S9-mix in all experiments. The positive controls induced appropriate increases in mutant frequency in all mutation experiments thus demonstrating the activity of the S9-mix and that the assay was performing satisfactorily in being capable of detecting known mutagens.

The test material is not mutagenic in L5178Y TK+/- cells treated in vitro in either the presence or absence of S9-mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test in rats, negative, according to OECD TG 474, Fox 2006

- In vivo unscheduled DNA synthesis, negative, according to OECD TG 486, Fox 2006

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA. All of the assays conducted showed negative results and it is concluded that the test material is not genotoxic. Metabolites of the test material did not induce revertants in four separate Ames tests, four separate mammalian cell gene mutation studies, three separate in vitro cytogenetic test studies and one rat micronucleus test. However, one in vitro cytogenetic test study recorded a positive genotoxic result for a test material metabolite.

 

Genetic toxicity in vitro

Ames

The test material was evaluated in a bacterial reverse mutation assay using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2 (pKM101) and WP2 uvrA (pKM101). This study was conducted in accordance with OECD TG 471 following GLP principles (Callander 2006). The investigations were performed with concentrations of 100, 200, 500, 1000, 2500, 5000 μg/plate in all experiments in the presence and absence of a rat liver-derived metabolic activation system (S9 mix).

In two independent experiments, the test material did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the strains used, either in the presence or absence of S9 mix. The sensitivity of the test system, and the metabolic activity of the S9 mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

The test material gave a negative (non-mutagenic) response in both the presence and absence of S9 mix.

 

The other Ames test, all performed according to OECD TG 471 and under GLP, are supporting studies (Sokolowski 2008 & 2010). Both studies tested concentrations up to 5000 µg/plate in S. typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA (pKM 101), and WP2 (pKM 101). Toxic effects were observed in the higher concentrations, but no substantial increase in revertant colony numbers were observed, neither in the presence or absence of metabolic activation (S9 mix).

 

In vitro chromosome aberration study in mammalian cells

The test material was evaluated for its clastogenic potential in an in vitro cytogenetic assay using human lymphocytes in two independent experiments treated in the presence and absence of a rat liver-derived metabolic activation system (S9 mix) (Fox 2006). This study was conducted in accordance with OECD TG 473 following GLP guidelines. In Experiment 1, cultures were treated for a period of 3 hours both in the presence and absence of S9 mix. In Experiment 2 cultures were treated for a period of 3 hours in the presence of S9 mix and 20 hours in the absence of S9 mix. All cultures were harvested 68 hours after culture initiation. The concentrations were chosen to be 20, 30, 50 μg/ mL (+S9 -mix) and 20, 30, 40 μg/ mL (-S9 -mix) in Experiment 1, and 20, 30, 50 μg/ mL (+S9 -mix) and 10, 15, 20 μg/ mL (-S9 -mix) in Experiment 2. Cultures were treated with the test material at appropriate concentrations for chromosomal aberration analysis along with the appropriate solvent and positive control cultures.

The highest concentrations selected for chromosome aberration analysis were based on reductions in mitotic activity. Concentration related reductions in mitotic activity were observed in cultures from both experiments, thus demonstrating that the test material is biologically active in this test system. In Experiment 1, there were small but statistically significant increases in the percentage of aberrant cells in the absence of S9 mix at 20 μg/mL and 30 μg/mL There were no increases in the percentage of aberrant cells at 40 μg/mL and the increases at 20 μg/mL and 30 μg/mL were within the historical control solvent control range for this laboratory. No other statistically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded. The sensitivity of the test system, and the metabolic activity of the S9 mix, were demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents.

The test material is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9 mix.

 

The other in vitro chromosome aberration study in mammalian cells, also performed according to OECD TG 473 and GLP, is a supporting study (Bohnenberger 2008). In this study, no clastogenic effects were observed in human lymphocytes exposed to concentrations up to 90.5 µg/mL in presence of S9 mix or 16.0 µg/mL in absence of S9 mix.

 

In vitro gene mutation assay in mammalian cells

To assess the potential of the test material to cause gene mutation or clastogenic effects in mammalian cells, L5178Y TK+/- mouse lymphoma cells were treated in vitro with various concentrations of the test substance, both in the presence and absence of a rat liver derived auxiliary metabolic system (S9 mix) (Clay 2006). This study was conducted in accordance with OECD TG 476 following GLP principles. Large and small mutant colonies were scored for all cultures in each experiment. Mutant frequencies were assessed by cell growth in the presence of trifluorothymidine after a 48 hour expression time. The test material was tested up to maximum concentrations of 40 μg/mL and 25 μg/mL in the presence and absence of S9 mix, respectively, in 3 independent experiments.

Minimum survival levels, compared to the solvent control cultures, of 14 % and 19 % were observed in cultures treated with the maximum concentrations of the test material in the presence and absence of S9 mix, respectively. No significant increases in mutant frequency were observed in cultures treated with the test material in either the presence or absence of S9 mix in all experiments. The positive controls induced appropriate increases in mutant frequency in all mutation experiments thus demonstrating the activity of the S9 mix and that the assay was performing satisfactorily in being capable of detecting known mutagens.

The test material is not mutagenic in L5178Y TK+/- cells treated in vitro in either the presence or absence of S9 mix.

 

The other in vitro gene mutation assay in mammalian cells, also performed according to OECD TG 476 and GLP, is a supporting study (Wollny 2008). In this study, no mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation were observed.

 

Genetic toxicity in vivo

In vivo micronucleus test

The test material has been evaluated for its ability to induce micronucleated immature erythrocytes in the bone marrow of HsdRCCHan: WIST rats in a study according to OECD TG 474 following GLP principles (Fox 2006). A single oral dose was given to groups of male rats at a dose level of 2000 mg/kg. This dose level is the limit dose level of the assay. Bone marrow samples were taken 24 and 48 hours after dosing.

No significant increases in the incidence of micronucleated immature erythrocytes, over the vehicle control values, were seen at either of the sampling times investigated. Comparison of the percentage of immature erythrocytes showed no significant differences at either of the sampling times between the vehicle control animals and those treated with the test material. The test system positive control, cyclophosphamide, induced biologically meaningful increases in

micronucleated immature erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the conditions of test, the test material is not clastogenic in the rat bone marrow micronucleus test.

 

In vivo unscheduled DNA synthesis assay

The test material was been evaluated, using an autoradiographic technique, for its ability to induce unscheduled DNA synthesis (UDS) in the liver of HsdRCCHan: WIST rats in a study according to OECD TG 486 following GLP principles (Fox 2006). A single oral dose was given to a group of male rats at a dose level of 2000 mg/kg. This dose level is the limit dose level of the assay. Two sampling times, 2 hours and 16 hours post-dose were used.

The values recorded for the mean net nuclear grain counts and the percentage of cells in repair clearly show that the test material did not induce DNA repair, as measured by UDS, at either time point investigated. The test system positive control, N-nitrosodimethylamine (N-DMA), induced marked increases in UDS, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known genotoxin.

Under the conditions of test, the test material did not induce DNA repair, as measured by unscheduled DNA synthesis, in the rat liver in vivo.

Justification for classification or non-classification

Based on the available data, classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.