pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +S9 negative, -S9 negative, S. typhimurium: TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA, OECD 471, Hertner 1991

- In vitro mammalian cell gene mutation test (HPRT), +S9 negative, -S9 negative, Chinese hamster lung fibroblasts (V79), OECD 476, Geleick 1991

- In vitro mammalian chromosome aberration test (CA), +S9 negative, -S9 negative, Chinese hamster ovary (CHO)/CCL61 cells, OECD 473, Geleick 1991

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:

2 Jul 1991 to 12 Aug 1991

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:

Adopted May 1983

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1987

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: THE MINISTRY OF HEALTH AND WELFARE, Japan , Guidelines of Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs (Part 1), Notification No. 118 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare.

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

Range in the (preliminary) cytotoxicity test (with and without microsomal activation): 20.6, 61.7, 185, 555, 1666 and 5000 μg/plate
Range in the mutagenicity test (with and without microsomal activation): 312, 625, 1250, 2500 and 5000 μg/plate
Justification for top dose: 5000 μg/plate is the maximum recommended dose according to the test guideline

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-Aminoanthracene (2-AA); 4-Nitroquinoline (4-NQ)

Details on test system and experimental conditions:

PRELIMINARY CYTOTOXICITY ASSAY: A preliminary toxicity test was carried out with strains s. typhimurium TA 100 and E.coli WP2uvrA with and without microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was the highest applicable concentration (due to the solubility limit of the test substance in the solvent) or 5000 μg/plate. The five lower concentrations decreased by a factor of 3 (The number of concentrations tested and the factor of dilution were in deviation to the SOP effective at the time of this study). The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

METHOD OF APPLICATION: in agar (plate incorporation)

PROTOCOL
- Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.
- 0.1 mL of the overnight cultures were mixed with 2 ml of top agar, either 0.5 mL of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 mL of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.
- The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and with the Escherichia coli strain WP2uvrA without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance (312 to 5000 μg/plate), a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied was determined in the preliminary toxicity test and the four lower concentrations were each decreased by a factor of 2. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.
- Colonies were counted electronically with an Artek counter. The results were sent online to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director

CRITERIA FOR A POSITIVE RESPONSE:
The test substance is considered to be mutagenic in this test system if one or both of the following conditions are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: s. typhimurium TA 98, TA 1535, TA 1537 and E. Coli WPuvrA.
-A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strains. typhimurium TA 100.
- Generally a concentration-related effect should be demonstrable

Statistics:

None.

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA100, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 50 and 25 mg/mL precipitates were visible. The highest concentration soluble in dimethylsulfoxide was found to be 12.5 mg/mL. The highest concentration tested was 5 mg/mL.

RANGE-FINDING/SCREENING STUDIES:
Six concentrations of the test substance ranging from 20.6 to 5000 μg/plate were tested with strains S. typhimurium TA 100 and E.coli WP2uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 μg/plate without activation and 5000 μg/plate with activation.

NUMBER OF CELLS WITH REVERSE MUTATION
In the original experiment performed without and with microsomal activation, none of the tested concentrations led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control. In the confirmatory experiment performed without and with microsomal activation, again, the tested concentrations did not lead to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control.

HISTORICAL CONTROL DATA
- Positive and negative historical control data can be found in Table 1 and 2 in ‘any other information on results incl. tables’. The data is presented as Arithmetic Mean (m) and Standard Deviation (s) of colony counts obtained in 91 separate experiments over the period of January 15, 1990 to January 02, 1991. Acceptable range for mean colony counts (=mean of values) of spontaneous revertants.

Table 1. Historical data positive and negative control in absence of metabolic activation.

Strain	Substance	µg/plate	Mean	StDev	Acceptable range
TA 100	Negative control		146.9	28.2	8-220
	sodium azide	2.0	762.9	257.2
TA 1535	Negative control		11.4	3.3	7-30
	sodium azide	2.0	699.4	277.8
E. Colie WP2uvrA	Negative control		20.2	4.5	8-40
	4-nitroquinoline-N-oxide	1.0	501.7	323.4
TA 98	Negative control		19.1	4.4	12-50
	2-nitrofluorene	10.0	1287.5	308.9
TA 1537	Negative control		7.9	2.6	3-20
	9-amino acridine	150.0	2158.9	583.3

Table 2. Historical data positive and negative control in presence of metabolic activation.

Strain	Substance	µg/plate	Mean	StDev	Acceptable range
TA 100	Negative control		142.2	25.8	70-220
	2-aminoanthracene	2.5	1768.8	544.2
TA 1535	Negative control		12.6	3.2	7-35
	Cyclophosphamide	400.0	349.6	126.1
E. Colie WP2uvrA	Negative control		22.3	5.2	8-50
	2-aminoanthracene	50.0	1109.7	277.1
TA 98	Negative control		35.4	5.7	20-70
	2-aminoanthracene	2.5	1759.9	470.3
TA 1537	Negative control		12.0	4.3	5-30
	2-aminoanthracene	5.0	204.3	82.4

Conclusions:

In this GLP compliant study, performed according to OECD 471, the test substance and its metabolites did not induce gene mutations in the tested strains of S. typhimurium and E. coli

Executive summary:

In this GLP compliant OECD 471 study (a reverse gene mutation assay in bacteria), the test substance in dimethylsulphoxide (DMSO) was tested on four histidine-auxotrophic strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and on the tryptophan-auxotrophic strain WP2uvrA of Escherichia coli, by plate incorporation, at concentrations of 0 (solvent control), 312.5, 625, 1250, 2500 and 5000 µg/plate. Two independent assays were performed, both experiments with and without S9 metabolic activation (S9 fraction from Arochlor 1254 induced rat liver). After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting with an Artek counter and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments. In the range finding test, normal background growth occurred with both strains, with or without metabolic activation and no appreciable cytotoxicity was observed at 5000 µg/plate, the highest concentration evaluated. In the main study, the test substance did not increase the number of revertants in any of the strains used, with or without metabolic activation, in either experiment when compared to the vehicle controls. There was no evidence of toxicity to the bacteria at any concentration, in either experiment. The positive controls produced a marked increase of the number of revertant colonies. Therefore, it was concluded that the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used in the study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Aug 1991 to 23 Oct 1991

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:

Adopted May 1984

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

Adopted May 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC: Mutagenicity testing and screening for carcinogenicity-In vitro mammalian cell gene mutation test: Official Journal of the European Comm. No L 133 61-63 (1988)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Number of passages if applicable: maximal 12
- Methods for maintenance in cell culture if applicable: Large stocks of the V79 cell line are stored in liquid nitrogen allowing the repeated use of the same cell culture batch in experiments. The cell culture was passaged weekly in low number (about 5000 cells per 175 cm2 flask) to keep the level of spontaneous mutants low and to prevent the cells from reaching a stationary phase of cell -growth. Twice a week the growth medium was replaced. After twelve passages the cells were replaced by a freshly thawed culture from our frozen stock.
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22 ± 1.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Seeding was done
in 25 ml growth medium (Ham's F10, 10% pre-tested foetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin) in 175 cm2 tissue-culture (plastic) flasks. The humidity in the incubator was adjusted to> 85% RH, the air was enriched to 5 ± 2% CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, using the Hoechst-day staining.

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal fraction S9 of Aroclor 1254-induced Tif: RAIf (SPF) rats

Test concentrations with justification for top dose:

PRELIMINARY CYTOTOXICITY TEST (RANGE FINDING TEST): Twelve concentrations ranging from 0.16 to 333.3 μg/mL separated by a factor of 2 were used. (with and without metabolic activation). The highest value was selected based on solubility of the test substance.
MUTAGENICITE TEST (MAIN EXPERIMENT): 5.21, 20.83, 83.33 and 333.33 μg/mL (with and without metabolic activation). The highest dose was selected based on the range finding study.

Vehicle / solvent:

Dimethylsulfoxide (DMSO) The final concentration of dimethylsulfoxide in the culture medium was 1%.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: N-nitroso-dimethylamine, 1.0 µL/mL (DMN), with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 2.5 - 5.0 x 10^6 cells

DURATION
- Preincubation period: seeded and incubated overnight
- Exposure duration: 5 hours (+S9) and 21 hours (-S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 to 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 15 days (+S9) and 15 to 16 days (-S9)

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (dimethylsulfoxide) control. The assays were performed in duplicates.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were fixed with methanol and stained with Giemsa stain.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: was estimated at the end of the expression period.

Evaluation criteria:

Criteria for a positive response
The test substance will be considered mutagenic in this test system, if either:
The mutant frequency of the treated culture exceeds that of the solvent controls by a mutant factor of 2.5 and there is a dose-dependent increase of the mutant frequency.
OR;
The mutant frequency in a treated culture exceeds that of the solvent control by a mutant factor of 3.0 at any concentration tested and reported and the absolute number of clones in the treated and untreated cultures differ by more than 20 clones per 10^6 cells plated.

Criteria for a negative response
The test substance will be considered to be inactive in this test system:
If there is no concentration-dependency of the mutant frequency values determined and the highest mutant frequency of a treated culture exceeds that of the solvent controls by a factor lower than 3.0 or the absolute number of clones in the treated and untreated culture with the highest mutant frequency value differs by less than 20 clones per 106 cells plated.
OR;
If there is a concentration-dependency of the mutant frequency values determined and the mutant frequency in a treated culture exceeds that of the solvent controls only by a factor lower than 2.5.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The highest concentration of test substance was determined in a preliminary solubilisation test to be 33.33 mg/mL soluble in DMSO. The highest concentration tested was 333.33 µg/mL. There was no precipitation of the test substance.

PRELIMINARY CYTOTOXICITY TEST (RANGE FINDING TEST):RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, the viability of the cells treated in the experiment with microsomal activation for 5 hours with the test substance was not reduced to a value lower than 10% of the negative control (dimethylsulfoxide) up to the highest applicable concentration of 333.33 μg/mL which represents the solubility limit of the test substance. In the experiment without activation the viability of the cells treated for 21 hours with the test substance was not reduced to a value lower than 10% of the negative (dimethylsulfoxide) control up to the highest applicable concentration of 333.33 μg/mL. Therefore, the original mutagenicity test with microsomal activation was performed with concentrations ranging from 5.21-333.33 μg/mL and the original mutagenicity test without microsomal activation was performed with concentrations ranging from 5.21-333.33 μg/mL.

NUMBER OF CELLS WITH MICRONUCLEI
Detailed description of test results of the original mutagenicity test
In the original mutagenicity test with microsomal activation, after screening with 6-thioguanine the mutant frequencies in the solvent controls were <4.00x10^-6 and 4.44x10^-6. The mean value used for calculation is 4.22x10^-6. At the lowest concentration tested, the calculated mutant frequency values were <4.00x10^-6 and 7.73x10^-6 . Comparison of this value with the mean of the two solvent controls revealed mutant factors of 1.00 and 1.83. At the higher concentrations up to the highest, the calculated mutant-frequency values were <4.00x10^-6 (factor 1.00) and 4.04x10^-6 (factor 1.00), 22.60x10^-6 (factor 5.35) and <4.00x10^-6 (factor 1.00), <4.00x10^-6 (factor 1.00) and <4.00x10^-6 resulting in a mutant factor of 1.00. Although the mutant factor at the concentration of 83.33μg/mL is higher than a factor 3 the absolute number of clones in the treated and untreated culture differs by less than 20 clones. Furthermore the result is not reproducible in the duplicate culture nor in the confirmatory experiment and therefore regarded as biologically insignificant. The positive controls treated with 1 µLDMN/mL medium revealed a mean mutant-frequency value of 708.69x10^-6, giving a mutant factor of 167.85.

The mutant-frequencies in the solvent controls of the cultures without microsomal activation were 5. 92x10^-6 and <4.00X10^-6 . The mean value used for calculation is 4.96x10^-6. At the lowest concentration tested, the calculated mutant frequency values were both <4.00x10^-6. Comparison of this value with the mean of the two solvent controls revealed mutant factors of 1.00. At the higher concentrations up to the highest, the calculated mutant-frequency values were 4.78x10^-6 (factor 1. 00) and 6.33x10^-6 (factor 1.28), <4.00x10^-6 (factor 1.00) and 5.37x10^-6 (factor 1.08), 4.62x10^-6 (factor 1.00) and <4.00x10^-6 resulting in a mutant factor of 1.00. The positive controls treated with 300 nL ethylmethanesulfonate/mL medium gave a mean mutant-frequency value of 2597.63x10^-6 and a corresponding mutant factor of 523.66.

Detailed description of the test results of the confirmatory mutagenicity test
In the confirmatory mutagenicity test with microsomal activation, after screening with 6-thioguanine the mutant frequencies in the solvent controls were 4.30x10^-6 and <4.00x10^-6. The mean value used for calculation is 4.15x10^-6. At the lowest concentration tested, the calculated mutant frequency values were both <4.00x10^-6. Comparison of this value with the mean of the two solvent controls revealed mutant factors of 1.00. At the higher concentrations up to the highest, the calculated mutant frequency values were <4.00x10^-6 (factor 1.00) and <4.00x10^-6 (factor 1.00), <4.00x10^-6 (factor 1.00) and <4.00x10^-6 (factor 1.00), 5.70x10^-6 (factor 1.37) and <4.00x10^-6 resulting in a mutant factor of 1.00.

The positive controls treated with 1 µLDMN/mL medium revealed a mean mutant-frequency value of 494.98x10^-6, giving a mutant factor of 119.28. The mutant-frequencies in the solvent controls of the cultures without microsomal activation were both <4.00x10^-6. This value represents the lower limit of sensitivity of this test system. The mean value used for calculation is 4.00x10^-6.
At all concentrations tested, the calculated mutant frequency values were <4.00x10^-6 resulting in a mutant factor of 1.00. The positive controls treated with 300 nL ethylmethanesulfonate/mL medium gave a mean mutant frequency value of 1397.50x10^-6 and a corresponding mutant factor of 349.37.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive and negative historical control data can be found in Table 1 in ‘any other information on results incl. tables’. The data is presented as Arithmetic Mean and Standard Deviation (s) of mutant frequencies (in x10^-6). Historical data is accumulated from April 1988 to December 1990.
 
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- An overview of the cytotoxicity ranges as percentage of negative control can be found in Table 2 to 5 in ‘any other information on results incl. tables’.

Table 1. Historical data from gene mutation tests with Chinese Hamster Cells V79.

Experiments with microsomal activation		Experiments without microsomal activation
Negative control	Positive control DMN 1.0 µL/mL	Negative control	Positive control EMS 0.3 µL/mL
8.7	255	<4.0	1365
<4.0	211	<4.0	2615
7.9	111	<4.0	1097
4.3	161	4.4	1803
<4.0	63	<4.0	841
4.6	117	<4.0	852
<4.0	170	5.9	1090
5.5	126	4.7	1314
<4.0	192	<4.0	1742
4.7	317	<4.0	1327
<4.0	102	<4.0	841
<4.0	166	<4.0	996
7.7	124	6.5	1588
<4.0	56	<4.0	966
6.0	175	<4.0	947
<4.0	95	<4.0	924
20.9	206	16.3	1779
<4.0	79	<4.0	1594
8.1	216	11.2	1322
<4.0	134	<4.0	943
<4.0	328	<4.0	1268
<4.0	269	<4.0	845
<4.0	113	<4.0	1629
<4.0	144	<4.0	1187
<4.0	113	<4.0	1629
4.0	144	<4.0	1187
<4.0	100	<4.0	542
<4.0	73	<4.0	935
<4.0	100	<4.0	542
<4.0	73	<4.0	935
<4.0	128	4.0	891
<4.0	69	4.5	837
4.8	137	<4.0	1607
5.1	126	<4.0	435
<4.0	131	<4.0	1184
<4.0	268	<4.0	1180
<4.0	94	<4.0	643
5.9	106	7.1	1813
8.3	424	11.5	1876
<4.0	121	<4.0	1162
5.6	96	<4.0	1105
<4.0	268	<4.0	1180
<4.0	103	<4.0	887
<4.0	226	<4.0	2770
<4.0	65	<4.0	811
13.6	102	11.3	581
<4.0	65	<4.0	811
13.7	102	11.3	581
5.0	61	5.9	1034
<4.0	255	<4.0	696
<4.0	117	<4.0	782
<4.0	177	<4.0	1211
<4.0	108	<4.0	607
4.2	236	8.2	1489
8.8	132	5.2	478
<4.0	86	<4.0	879
<4.0	157	5.3	1024
6.3	151	8.4	730
<4.0	215	<4.0	1057
<4.0	123	<4.0	930
4.2	47	<4.0	626
4.8	122	<4.0	821
4.9	179	<4.0	814
32.9	287	19.1	1391
This experiment
4.2	709	5.0	2598
4.2	495	<4.0	1398

Table 2. Cytotoxicity determined after treatment experiment with microsomal activation (original experiment).

Treatment		Mean of survivor I clones	Number of viable cells (x10^6)	Acute cytotoxicity (% of control)
Negative control		85.67	7.17
		67.50	5.70
Positive control (DMN)	1 µg/mL	57.17	5.18	19.54
	1 µg/mL	35.83	2.83	55.95
Test substance	333.33 µg/ml	72.17	5.59	13.15
	333.33 µg/ml	39.83	1.51	76.57
	83.33 µg/ml	43.00	2.23	65.36
	83.33 µg/ml	43.33	2.44	62.12
	20.83 µg/ml	57.67	4.17	35.14
	20.83 µg/ml	75.00	6.07	5.57
	5.21 µg/ml	73.17	5.25	18.39
	5.21 µg/ml	59.00	4.50	30.11

 

Table 3. Cytotoxicity determined after treatment experiment without microsomal activation (original experiment).

Treatment		Mean of survivor I clones	Number of viable cells (x10^6)	Acute cytotoxicity (% of control)
Negative control		76.50	12.69
		89.33	8.79
Positive control (EMS)	300 nL/mL	34.00	4.71	56.18
	300 nL/mL	41.33	5.31	50.59
Test substance	333.33 µg/ml	98.50	9.79	8.88
	333.33 µg/ml	98.67	8.10	24.62
	83.33 µg/ml	84.17	5.91	44.93
	83.33 µg/ml	84.83	10.97	n.t.
	20.83 µg/ml	79.50	7.28	32.24
	20.83 µg/ml	75.17	11.33	n.t.
	5.21 µg/ml	81.83	9.67	9.96
	5.21 µg/ml	106.00	8.98	16.38

n.t.: not toxic

 

Table 4. Cytotoxicity determined after treatment experiment with microsomal activation (confirmatory experiment).

Treatment		Mean of survivor I clones	Number of viable cells (x10^6)	Acute cytotoxicity (% of control)
Negative control		110.00	8.46
		107.33	8.89
Positive control (DMN)	1 µg/mL	54.67	3.78	56.40
	1 µg/mL	63.00	5.24	39.54
Test substance	333.33 µg/ml	54.33	3.82	56.00
	333.33 µg/ml	58.00	4.19	51.66
	83.33 µg/ml	67.00	5.38	37.93
	83.33 µg/ml	72.33	6.24	28.04
	20.83 µg/ml	81.00	7.23	16.67
	20.83 µg/ml	71.67	6.66	23.27
	5.21 µg/ml	83.00	6.84	21.16
	5.21 µg/ml	78.00	6.76	22.10

 

 

Table 5. Cytotoxicity determined after treatment experiment without microsomal activation (confirmatory experiment).

Treatment		Mean of survivor I clones	Number of viable cells (x10^6)	Acute cytotoxicity (% of control)
Negative control		157.67	14.29
		145.67	12.57
Positive control (EMS)	300 nL/mL	95.67	6.69	50.17
	300 nL/mL	90.00	7.81	41.85
Test substance	333.33 µg/ml	145.00	12.24	8.84
	333.33 µg/ml	167.00	17.18	n.t.
	83.33 µg/ml	173.33	17.45	n.t.
	83.33 µg/ml	163.33	17.69	n.t.
	20.83 µg/ml	126.00	12.73	5.24
	20.83 µg/ml	140.33	13.53	n.t.
	5.21 µg/ml	126.33	14.88	n.t.
	5.21 µg/ml	129.67	14.61	n.t.

n.t.: not toxic

Conclusions:

In this GLP compliant study, performed according to OECD TG 476, the test substance or its metabolites did not induce gene mutations in cultured V79 Chinese hamster cells with or without metabolic activation.

Executive summary:

In a GLP compliant OECD 476 study Chinese hamster V79 cell cultures were exposed to the test substance with or without metabolic activation. The highest concentrations used in the experiments were determined in an initial cytotoxicity range-finding test in which 12 concentrations of the test substance, from 0.16 to 333.3 mg/mL (limit of solubility), were tested with and without S9 (Aroclor 1254-induced Tif: RAIf (SPF) rats) metabolic activation. In the main study, the test substance in DMSO (final concentration 1%) was tested at concentrations of 0 (solvent control), 5.21, 20.83, 83.33 and 333.33 μg/mL with and without S9 metabolic activation. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment). In each assay, cultures were treated in duplicate, a positive and a negative (dimethylsulfoxide) control for 5 hours with S9 and for 21 hours without S9. Ethylmethansulphonate at a concentration of 300 nL/mL and the promutagen N-nitroso-dimethylamine at a concentration of 1.0 μL/mL were used as positive controls. After exposure, the cultures were incubated at 37°C for one week during which the cells could recover and divide to express the mutant phenotype. After 2.5 days the cultures were subcultured for cytotoxicity testing. At the end of the expression period the cultures were counted with a haemocytometer or Coulter counter. The mutagenicity cultures were exposure to 6-thioguanine for mutant selection by incubation at 37°C for 7-8 days, after which the cultures were fixed and stained and the mutated clones counted by eye. Cytotoxicity was estimated from the cloning efficiency immediately after the recovery period for determination of survival values. Viability at the end of expression was estimated from the cloning efficiency. In the preliminary cytotoxicity test, the highest concentration with and without metabolic activation produced 10% growth inhibition. In both experiments, with and without metabolic activation, no significant increase in the mutation frequencies was observed following exposure to the test substance. In contrast, the positive control substances DMN and EMS produced highly statistically significant increases in mutation frequencies. The test substance or its metabolites did not induce gene mutations in cultured V79 Chinese hamster cells either in the presence or absence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Aug 1991 to 1 Nov 1991

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)

Version / remarks:

Adopted May 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Version / remarks:

Adopted May 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 19, 1984/June 1989

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

MAFF Japan (January 1985)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CCL 61

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: This test system permits the detection of structural chromosome aberrations in Chinese hamster ovary cells in vitro induced by the test substance or by its metabolites

MEDIA USED
The cell line CCL 61 (Chinese hamster ovary cells) was maintained in culture medium consisting of Nutrient Mixture F-12 supplemented with 10% fetal calf serum+ Penicillin/Streptomycin 100 units/mL / 100 μg/ mL.

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction of Acolor 1254-induced male rats (Tif: RAIf (SPF)) + cofactors

Test concentrations with justification for top dose:

JUSTIFICATION FOR TOP DOSE: The highest concentration was the solubility limit and caused up to 27.3 % suppression of mitotic activity

CYTOTOXICITY/MUTAGENICITY TEST
Original study: 2.58, 5.16, 10.31, 20.63, 41.25, 82.5, 165.0 and 330 µg/mL (with and without metabolic activation)
Confirmatory study: experiment 1 and 2; 20.63, 41.25, 82.5, 165.0 and 330 µg/mL (with and without metabolic activation). Experiment 3 and 4; 2.58, 5.16, 10.31, 20.63, 41.25, 82.5, 165.0 and 330 µg/mL (with and without metabolic activation).

CHROMOSOME ABBERATION STUDY
Original study: 82.5, 165 and 330 μg/mL (with and without metabolic activation)
Confirmatory study: 82.5, 165 and 330 μg/mL (with and without metabolic activation)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 29 hours
- Experimental design
Experiment 1, 18 hours incubation time, no expression time before harvesting, original study + confirmatory study, without metabolic activation.
Experiment 2, 3 hours incubation time, 15 hours expression time before harvesting, original study + confirmatory study, with metabolic activation.
Experiment 3, 42 hours incubation time, no expression time before harvesting, confirmatory study, without metabolic activation.
Experiment 4, 3 hours incubation time, 39 hours expression time before harvesting, confirmatory study, with metabolic activation.

SPINDLE INHIBITOR: Colcemide, 0.4 µg/mL. Added two hours before harvesting of the cells.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION: The experiment was terminated by hypotonic treatment (0.075 M KCl solution) of the cells, followed by fixation (methanol: acetic acid, 3:1). Drop preparations were made by the air-drying technique.

SELECTION OF CONCENTRATIONS FOR ANALYSIS: The clastogenic activity was assessed at concentrations of 82.5, 165 and 330 μg/ml without and with metabolic activation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: Two hundred metaphases whenever possible were examined from the vehicle control and from the cultures treated with the various concentrations of the test substance. At least fifty metaphases each from the appropriate positive controls were analysed.

SCORING OF THE SLIDES
Prior analysis the selected slides were coded, likewise the cultures treated with the vehicle alone as well as the positive control. Whenever possible two hundred metaphase figures with 19 to 21 centromeres from cultures of two falcon flasks in the vehicle control, in the treated groups and at least fifty metaphases in the positive controls were examined for the following aberrations:
a) specific fragments, aberrations: breaks, exchanges, deletions
b) unspecific aberrations: gaps and chromosome decay
c) numerical alterations (metaphases with >21 centromeres) will be registered, yet reported only in case of deviations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE: The test substance is generally considered to be active in the Chinese Hamster cells if the following conditions are met. The percentage of specific aberrations in a treatment group is higher than 6.0 and differs statistically significant from the respective value of the negative control. And a concentration-related response should be demonstrable.

CRITERIA FOR A NEGATIVE RESPONSE: The test substance is generally considered to be inactive in the Chinese Hamster cells if the following conditions are met. The percentage of specific aberrations in all treatment groups is less than or equal to 6.0 and does not differ statistically significant from the respective value of the negative control.

Statistics:

The evaluated numbers of specific aberrations were subjected to statistical analysis. Tests were performed based upon the presence of any specific aberration. In a preliminary test the data were tested for flask effects (dependence of cells within each flask) using a chi-square test. The nonsignificant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly, a chi-squared test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CCL 61

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The highest concentration of the test substance soluble in DMSO (stock solution) was 33.3 mg/mL. The highest concentration tested was 330 μg/mL. There was no precipitation of the test substance.

RANGE-FINDING/SCREENING STUDIES:
The cytotoxicity test was performed as an integral part of the mutagenicity test.

CHROMOSOME ABERRATION:
- The treatment of the cultures with mitomycin C, 0.2 μg/mL and cyclophosphamide, 40.0 μg/mL, respectively, was followed by a high incidence of specific chromosomal aberrations in the experiments one and two of the original study (44 % and 43 %) and in the experiments one and two of the confirmatory study (40 % and 38 %). These values differed statistically significant from their respective negative control value.
- Unspecific chromosomal aberrations in the form of chromatid gaps found in all experiments were within the frequency generally observed.
- None of the observed aberration frequencies differed statistically significant from their respective negative control value.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: no data
- Negative (DMSO) historical control data: an overview with the historical ranges for negative controls can be found in Table 1 in ‘any other information on results incl. tables’.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The highest concentration of 330 µg/mL selected for analysis in the first experiment of the original study caused 27.3% suppression of mitotic activity. The highest concentration of 330 μg/mL selected for analysis in the second experiment of the original study caused no suppression of mitotic activity. In the third experiment of the confirmatory study with a 42 hours treatment period the highest concentration of 330 µg/mL selected for analysis caused no suppression of mitotic activity. In the fourth experiment (3 hours treatment / 39 hours recovery) the highest concentration of 330 µg/mL selected for analysis caused 9.2% suppression of mitotic activity. The concentration of 330 µg/mL selected as the highest scorable concentration in all experiments represents the solubility limit of the test substance.
 

ANALYSIS OF TEST SUBSTANCE: The recovery was determined as 107.7%

 Table 1. Historical data range for the negative control (DMSO) for different treatment durations.

	18 hours treatment / no recovery		3 hours treatment / 15 hours recovery		42 hours treatment / no recovery		3 hours treatment / 39 hours recovery
	100 metaphases	200 metaphases	100 metaphases	200 metaphases	100 metaphases	200 metaphases	100 metaphases	200 metaphases
Metaphases with specific aberrations (in %)	0 – 7	0.5 - 3	0 – 3	0 - 3	1 - 6	0 - 4	1	0 - 3.5
Metaphases with unspecific aberrations (in %)	0 - 10	1.5 - 5	0 - 10	0 - 7	3 - 7	0.5 – 6.5	3 – 6	0 - 6

Conclusions:

In this GLP compliant study, performed according to OECD TG 473, there was no evidence of a clastogenic effect of the test substance in in vitro cultures of Chinese hamster ovary cells.

Executive summary:

In this GLP compliant OECD 473 study, the test substance was evaluated for clastogenic effects in Chinese hamster ovary cells (CHO CLL61 cells), by incorporation in the culture medium. The test substance was dissolved in dimethylsulphoxide (DMSO, final concentration of 1%). In the original studies (experiment 1 without metabolic activation and experiment 2 with metabolic activation) cells were exposed to 2.58, 5.16, 10.31, 20.63, 41.25, 82.5, 165.0 and 330 µg/mL test substance for 18 and 3 hours respectively. The same concentrations were used for confirmatory studies from experiment 3 (without metabolic activation) and 4 (with metabolic activation), in which cells were exposed for 42 and 3 hours respectively. For the confirmatory studies of experiment 1 and 2, cells were exposed to 20.63, 41.25, 82.5, 165.0 and 330 µg/mL test substance. For all experiments the selected concentrations for chromosome analysis were 82.5, 165 and 330 µg/mL. Higher concentrations of the test substance could not be achieved because of solubility limitations. Experiments with metabolic activation with an exposure time of 3 hours were followed by a recovery period of 15 hours (experiment 2 and 3) or 39 hours (experiment 4). Mitomycin C , 0.2 µg/mL, and cyclophosphamide, 40.0 µg/mL, were used as positive control to validate the sensitivity of the assay. Two hours prior to harvesting, the cultures were treated with 0.4 µg/mL colcemide to arrest cells in metaphase.

A cytotoxicity test was performed as an integral part of the study by determination of the percentage mitotic suppression in at least 2000 cells from one slide/group. Whenever possible two hundred metaphase figures with 19 to 21 centromeres from cultures of two falcon flasks in the vehicle control, in the treated groups and at least fifty metaphases in the positive controls were examined The slides were examined blind for specific and non-specific structural aberrations.

In the experiments performed without and with metabolic activation no biologically relevant increase in the number of metaphases containing specific chromosomal aberrations was observed. None of the observed aberration frequencies differed statistically significant from their respective negative control value. Unspecific chromosomal aberrations in the form of chromatid gaps found in all experiments were within the frequency generally observed. Treatment of CHO cells with the positive control materials produced a high incidence of specific chromosomal aberrations (38% to 44%), demonstrating the sensitivity of the test system. There was no evidence of a clastogenic effect of the test substance in in-vitro cultures of Chinese hamster ovary cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation in bacteria

In a reverse gene mutation assay in bacteria, the test substance (purity 98.0%) in dimethylsulphoxide (DMSO) was tested on four histidine-auxotrophic strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and on the tryptophan-auxotrophic strain WP2uvrA of Escherichia coli, by plate incorporation, at concentrations of 0 (solvent control), 312.5, 625, 1250, 2500 and 5000 µg/plate (Hertner 1991). Two independent assays were performed, both experiments with and without S9 metabolic activation (S9 fraction from Arochlor 1254 induced rat liver). After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting with an Artek counter and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments.

In the range finding test, normal background growth occurred with both strains, with or without metabolic activation and no appreciable cytotoxicity was observed at 5000 µg/plate, the highest concentration evaluated. In the main study, the test substance did not increase the number of revertants in any of the strains used, with or without metabolic activation, in either experiment when compared to the vehicle controls. There was no evidence of toxicity to the bacteria at any concentration, in either experiment. The positive controls produced a marked increase of the number of revertant colonies. The test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used in the study.

In vitro gene mutation in mammalian cells

In a GLP compliant OECD 476 study Chinese hamster V79 cell cultures were exposed to the test substance with or without metabolic activation (Geleick 1991). The highest concentrations used in the experiments were determined in an initial cytotoxicity range-finding test in which 12 concentrations of the test substance, from 0.16 to 333.3 mg/mL (limit of solubility), were tested with and without S9 (Aroclor 1254-induced Tif: RAIf (SPF) rats) metabolic activation. In the main study, the test substance in DMSO (final concentration 1%) was tested at concentrations of 0 (solvent control), 5.21, 20.83, 83.33 and 333.33 μg/mL with and without S9 metabolic activation. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment). In each assay, cultures were treated in duplicate, a positive and a negative (dimethylsulfoxide) control for 5 hours with S9 and for 21 hours without S9. Ethylmethansulphonate at a concentration of 300 nL/mL and the promutagen N-nitroso-dimethylamine at a concentration of 1.0 μL/mL were used as positive controls. After exposure, the cultures were incubated at 37°C for one week during which the cells could recover and divide to express the mutant phenotype. After 2.5 days the cultures were subcultured for cytotoxicity testing. At the end of the expression period the cultures were counted with a haemocytometer or Coulter counter. The mutagenicity cultures were exposure to 6-thioguanine for mutant selection by incubation at 37°C for 7-8 days, after which the cultures were fixed and stained and the mutated clones counted by eye. Cytotoxicity was estimated from the cloning efficiency immediately after the recovery period for determination of survival values. Viability at the end of expression was estimated from the cloning efficiency. In the preliminary cytotoxicity test, the highest concentration with and without metabolic activation produced 10% growth inhibition. In both experiments, with and without metabolic activation, no significant increase in the mutation frequencies was observed following exposure to the test substance. In contrast, the positive control substances DMN and EMS produced highly statistically significant increases in mutation frequencies. The test substance or its metabolites did not induce gene mutations in cultured V79 Chinese hamster cells either in the presence or absence of metabolic activation.

In vitro clastogenicity study in mammalian cells

In a GLP compliant OECD TG 473 study, the test substance (purity 98.0%) was evaluated for clastogenic effects in Chinese hamster ovary cells (CHO CLL61 cells), by incorporation in the culture medium (Hertner 1991). The test substance was dissolved in DMSO (final concentration of 1%). In the original studies (experiment 1 without metabolic activation and experiment 2 with metabolic activation) cells were exposed to 2.58, 5.16, 10.31, 20.63, 41.25, 82.5, 165.0 and 330 µg/mL test substance for 18 and 3 hours respectively. The same concentrations were used for confirmatory studies from experiment 3 (without metabolic activation) and 4 (with metabolic activation), in which cells were exposed for 42 and 3 hours respectively. For the confirmatory studies of experiment 1 and 2, cells were exposed to 20.63, 41.25, 82.5, 165.0 and 330 µg/mL test substance. For all experiments the selected concentrations for chromosome analysis were 82.5, 165 and 330 µg/mL. Higher concentrations of the test substance could not be achieved because of solubility limitations. Experiments with metabolic activation with an exposure time of 3 hours were followed by a recovery period of 15 hours (experiment 2 and 3) or 39 hours (experiment 4). Mitomycin C , 0.2 µg/mL, and cyclophosphamide, 40.0 µg/mL, were used as positive control to validate the sensitivity of the assay. Two hours prior to harvesting, the cultures were treated with 0.4 µg/mL colcemide to arrest cells in metaphase.

A cytotoxicity test was performed as an integral part of the study by determination of the percentage mitotic suppression in at least 2000 cells from one slide/group. Whenever possible two hundred metaphase figures with 19 to 21 centromeres from cultures of two falcon flasks in the vehicle control, in the treated groups and at least fifty metaphases in the positive controls were examined the slides were examined blind for specific and non-specific structural aberrations.

In the experiments performed without and with metabolic activation no biologically relevant increase in the number of metaphases containing specific chromosomal aberrations was observed. None of the observed aberration frequencies differed statistically significant from their respective negative control value. Unspecific chromosomal aberrations in the form of chromatid gaps found in all experiments were within the frequency generally observed. Treatment of CHO cells with the positive control materials produced a high incidence of specific chromosomal aberrations (38% to 44%), demonstrating the sensitivity of the test system. There was no evidence of a clastogenic effect of the test substance in in-vitro cultures of Chinese hamster ovary cells.

In vitro sister chromatid exchange assay

Test system: Primary hepatocytes were prepared by in situ collagenase perfusion of male rat (Tif:RAIf) liver (Hertner 1991). The hepatocytes were cultured at 37 °C in Williams' Medium E containing 10% fetal bovine serum and antibiotics. For the DNA repair tests, 4*E5 cells were seeded in 4 mL compartments of multi-hole plates containing gelatinised coverslips. The cells were allowed to attach during 1.5 to 2 hours. Unattached cells were then removed and the cultures were provided with fresh medium. After an adhesion period, the compartments were treated under each of the following conditions: test substance at concentrations of 0, 2.78, 8.33, 25, 75, 150 and 300 mg/mL, the positive control 2-Acetamino-fluorene (2-AAF), and a negative control containing the vehicle DMSO. Immediately after the addition of the test substance, 8 mCi/mL 3H-thymidine were added and the cells were incubated for 16 to 18 hours. Autoradiographs were prepared and stained in haematoxylin/eosine. In three slides per group a total of 150 nuclei were scored and the number of silver grains was determined. Two independent experiments were performed.

In the cytotoxicity test, the number of viable cells was decreased to 23% at 330μg/mL, whereas at 165μg/mL 49% of the cells were viable. Consequently, a concentration of 300μg/mL was considered as the highest suitable concentration still being in the cytotoxic range. In both, the original and confirmatory experiments comparison of the mean gross and the mean net values of silver grains per nucleus in the vehicle control and in the cultures treated with the various concentrations of the test substance again revealed no significant deviations. Likewise the percentage of nuclei in repair with respect to their gross and their net numbers of silver grains per nucleus after treatment with the test substance showed no significant increase if compared with the vehicle control.

The test substancewas not mutagenic under the conditions of this test. It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the test substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.

In vivo mammalian somatic cell study: cytogenicity - erythrocyte micronucleus

The micronucleus test in mice was conducted at single oral dose levels of 0, 200, 600 and 2000 mg/kg body weight on male and female mice (administration: 10 mL/kg bw via gavage) (Ogorek 1998). The high dose of 2000 mg/kg is currently the highest limit dose set in the appropriate guideline. The test compound was formulated in a mixture of aqueous CMC (0.5%) and Tween 80 (0.4%). The mouse strain was TifIbm:MAG. This strain is identical with the former Tif MAG f (SPF) mouse. An additional (positive control) group received 64 mg/kg cyclophosphamide and was sacrificed 24 hours after application. At all dose levels, groups of 5 male and 5 female mice were sacrificed at 24 and 48 hours after dosing. Femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for the presence of micronuclei, indicative of clastogenic and/or aneugenic effects.

At the high dose of 2000 mg/kg, two males out of 10 males were found dead on the day after treatment and were replaced by reserve animals. All other animals survived until scheduled necropsy. None of the animals showed signs of toxicity. In all groups exposed to the test substance assessed at the different time points after treatment, neither statistically significant nor biologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group. The positive control group showed a clear mutagenic effect.

It is concluded that there was no evidence of clastogenic or aneugenic effects in mice treated with the test substance in this study.

In vivo mammalian cell study: DNA damage and/or repair - UDS assay

The purpose of this study was to investigate whether the test substance induces unscheduled DNA synthesis (UDS) in vivo in the mouse liver (Clay 2006a). The test compound used in this study was dissolved in sterile, double deionised water, Nitrosodimethylamine dissolved in sterile, double deionised water served as positive control. Compounds and vehicle were orally dosed in a volume of 10 mL/kg bw, except for animals dosed with 2000 mg/kg bw, which received 20 mL/kg bw. Male B6C3F1 mice were used as test system and were allowed to acclimatise and randomised before the study commenced.

To determine the maximum tolerable dose (MTD), 3 animals were dosed with 2000, or 1250 mg/kg bw and observed for clinical signs or mortality for 4 days. After termination, liver was removed, fixed, and processed for histological evaluation to ensure that the MTD based on systemic toxicity did not induce excessive toxicity in the target organ.

In the main study, groups of 3 male mice were treated with vehicle, test compound (1st attempt: 2000 mg/kg bw; 2nd attempt: 625, and 1250 mg/kg bw) and positive control compound. 2 or 16 h post dosage, animals were killed by over-exposure to halothane and hepatocytes were isolated. Hepatocytes were allowed to attach on glass coverslips in Williams’ E complete medium for at least 90 min. Three cultures were prepared for each treatment. Then, medium was replaced with medium without serum and with 3H-thymidine. After 4 h medium was changed for non-radioactive medium and cells were incubated for additional 12 h. Then cells were fixed, two slides were processed for determination of autoradiography (the remaining third slide was held in reserve and was only processed if required). The coded slides were analysed for UDS induction with an electronic image analysis system. 50 cells from each slide were analysed. Mean nuclear grain count, mean cytoplasmic grain count, mean net nuclear grain count and the percentage of cells in repair for each slide and each animal were calculated.

An occurrence of mean net nuclear grain count of zero or above in a test substance treated animal is considered a positive response in this animal. Reproducibility of this response in animals treated concurrently and in an independent experiment was needed to conclude that the test compound was positive in UDS test. Results that were not reproducible or had only a slight effect (mean net nuclear grain count between –1 and 0) had to be given further investigation.

Tolerability test: No adverse clinical signs were observed for any animal in the group dosed with 2000 mg/kg bw. The MTD was therefore considered to be in excess of 2000 mg/kg bw. In the main group dosed with 2000 mg/kg bw one animal was found dead immediately prior to scheduled termination time (16 h post dose) and a further animal was killed for humane reasons after showing severe clinical signs including extreme subdued appearance, shaking, eyes half closed, reduced breathing rate and cold touch. The remaining 3 animals in this group showed involuntary twitching and slightly subdued appearance. Hence, the dose of 2000 mg/kg bw was considered in excess of MTD, all remaining animals were sacrificed and a further group of three animals was used to determine MTD. No adverse clinical signs were observed for any animal in the group dosed with 1250 mg/kg bw. Examination of the internal organs showed no treatment related macroscopic or microscopic findings in the liver in any of the animals used for MTD determination.

The main study: No adverse reactions were observed in test compound treated animals. Examination of internal organs showed no treatment related effects in these animals. Perfusion process for one treated animal failed for technical reasons, this animal was replaced by another treated animal. Hepatocytes prepared from all animals were examined microscopically. No apparent sings of excessive cytotoxicity were observed on slides from animals dosed with test compound. Test compound caused no increases, compared to the vehicle control, in mean net nuclear grain count, or in percentage of cells in repair, at either dose level or time point investigated. Hepatocytes from all test compound treated animals had mean net nuclear grain count of less than zero. The data indicate no evidence for induction of UDS by test compound. The positive control substance induced marked increases in the mean net nuclear grain counts and percentage of cells in repair, demonstrating the sensitivity of the test system.

Under the conditions of this study, the test substance did not induce DNA repair, as measured by unscheduled DNA synthesis, in the mouse liver in vivo. This result does not change the assessment of the test substance of not being mutagenic or genotoxic.

In vivo mammalian cell study: DNA damage and/or repair - comet assay

The test substance has been evaluated for its ability to induce DNA damage, as measured by the Comet assay, in the liver of B6C3F1 mice (Clay 2006b). The study was conducted in two phases. In the first phase the maximal tolerable dose was established as 1250 mg/kg. This dose and a lower dose (625 mg/kg) were used in phase II. In Phase II, groups of male mice (4 animals per group) were given a single oral dose of 625 and 1250 mg/kg. N-methyl-N-nitrosourea (MNU, 100 mg/kg) was used as positive control. Two sampling times, 2 and 16 hours post-dose were used. The left lobe of the liver was removed from each animals and made into a cell suspension. For each animal, three comet slides were prepared and 150 cells were assessed using a Comet Assay III system. Measurements made for each cell included head length, tail length, % head intensity, % tail intensity and tail moment.

The test substance induced no increases in mean tail intensity, compared to the vehicle controls, at either dose level tested at either sampling time investigated. The test system positive control, N-methyl-N-nitrosourea, induced increases in mean tail intensity, 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 substance produced a negative response in the Comet assay (for DNA damage) in the mouse liver in vivo.

In vivo mammalian somatic cell study: cytogenicity - erythrocyte micronucleus

A tolerability test was performed to select the dose levels for the mutagenicity experiments (Hertner 1991d). The tolerability test was performed with three groups of two mice (one female and one male) receiving one single application. One group received the highest applicable dose (5000 mg/kg bw/day) and the other two groups received the doses of 1/4 and 1/16 of that amount respectively.

In the first part of the test with the test substance 8 male and 8 female mice (Tif:MAGf (SPF)) were administered single oral doses of 4000 mg/kg bw/day and sacrificed 16, 24 and 48 hours thereafter.

In the second part of the study, single oral doses of 1000, 2000 and 4000 mg/kg bw/day were given and the animals (8 males and 8 females per group) were killed 24 hours later.

The sensitivity of the test system was checked by treating one further identically sized group of animals with the known mutagen cyclophosphamide. Animals of this group were killed 24 hours after treatment. In addition, negative control groups (arachis oil) were included in both parts of the test. After sacrifice, the bone marrow from both femur shafts was removed and smears were prepared on slides. The slides were examined scoring 1000 polychromatic erythrocytes per animal for the incidence of micronuclei.

Tolerability test: One out of two animals administered 5000 mg/kg bw/day died while the mice dosed with 1250 and 312.5 mg/kg bw/day survived. Based on this result, it was concluded that 5000 mg/kg exceeded the tolerated dose. All four mice administered 4000 mg/kg survived. Therefore this dose was selected as the highest dose for the micronucleus test.

Micronucleus-test, first part: The animals were treated once with 4000 mg/kg bw/day (as determined in the tolerability test) and sacrificed 16, 24 and 48 hours thereafter. In the 16 and 24 hours treatment groups, 3 males and 3 females died in each group. Within 48 hours 3 males and 2 females died. The evaluation of the bone marrow smears showed no statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at the respective sampling times. No increased incidence of micronucleated polychromatic erythrocytes (PCEs) was recorded after treatment with the test substance at any sampling time.

Micronucleus-test, second part: The animals were treated once with the doses of 1000, 2000 and 4000 mg/kg bw/day and sacrificed 24 hours thereafter. Mortality occurred at 1000 mg/kg bw/day in 1 male and 1 female, at 2000 mg/kg bw/day in 1 male and 2 females, and at 4000 mg/kg bw/day in 2 males and 2 females. The evaluation of the bone marrow smears from the animals treated with the test substance showed no statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with those from the animals of the negative control group.

 The test substance was not mutagenic in this test system. It is concluded that under given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance.

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.