N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic when observed in three in vitro studies (Ames test, HPRT test, in vitro MNT) in the presence or absence of a metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphocytes
Main Assay1: human whole blood (BioIVT (UK))
Main Assay2, 3: human whole blood (Biopredic International (France)), two batches (pooled)
- Normal cell cycle time (negative control): 1.5-2.0 cell cycle length

For lymphocytes:
- Sex, age and number of blood donors:
Main Assay 1 -1 donor: Female, Age 27 years old, (ERBC Code Number 2020/36)
Main Assay 2 - 2 donors: Male, Age 24 years old, (ERBC Code Number 2020/63), Female, Age 33 years old (ERBC Code Number 2020/64)
Main Assay 3 - 2 donors: Male, Age 23 years old, (ERBC Code Number 2021/11), Female, Age 23 years old (ERBC Code Number 2021/12)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: pooled
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

CULTURE MEDIUM
- RPMI 1640 1x (Dutch modification): 500mL
- Foetal Calf Serum: 100mL
- L-Glutamine (200mM): 6.25mL
- Antibiotic solution: 1.25mL

- foetal calf serum was heat-inactivated at 56°C for 20 minutes before use.
- For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used
in the following proportion: 10mL of PHA was added to 500mL of medium.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital – 5,6-Benzoflavone

Test concentrations with justification for top dose:

Details on the test concentrations are given in table in "Any other information on materials and methods incl. tables"

Main Assay 1: steep decline of cytotoxicity and no concentration tested showed an adequate cytotoxicity.
Main Assay 2: short treatment series were not consistent with those of Main Assay 1. Following the continuous treatment in the absence of S9 metabolism treatment, marked and moderate cytotoxicity was seen at the two highest dose levels tested of 25.3 and 22.0 μg/mL (64% and 41%, respectively).
Main Assay 3: following treatment in the absence of S9 metabolism, severe cytotoxicity was observed from 18.5 μg/mL onward where few cells or no cells were recovered onto slides.Following treatment in the presence of S9 metabolism, severe cytotoxicity was observed from 27.8 μg/mL onward where no cells were recovered onto slides.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent proved to be the best solvent and a clear solution was obtained

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (1%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: with S9 mix: 3h; without S9 mix: 3h and 31h
- Expression time (cells in growth medium): 31h (continous treatment, -S9), 32.5h (short treatment, +/-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): not specified

STAIN (for cytogenetic assays): Acridine Orange (0.1 mg/mL in PBS)

NUMBER OF REPLICATIONS:
Two replicate cell cultures were prepared at each test point.

NUMBER OF CELLS EVALUATED:
- selected doses, untreated, solvent controls, positive control (Cyclophosphamide): 1000 binucleated cells per culture were scored.
- cultures treated with colchicine: 1000 mononucleated cells per cell culture were scored.

DETERMINATION OF CYTOTOXICITY
CBPI was used to measure the cytotoxic effect.
CBPI = (mononucleated + 2xbinucleated + 3x multinucleated)/ total number of cells counted
%Cytotoxicity = 100-100(CBPIt -1/CBPIc-1), t= test item treated culture, c= solvent control culture


POSITIVE CONTROLS
- Without metabolic activation: Colchicine (batch no. SLBX1712, obtained from Sigma).
- With metabolic activation: Cyclophosphamide (labelled as: Cyclophosphamide monohydrate, batch no. MKCG5464, obtained from Sigma)
- All positive control items were dissolved immediately prior to use in sterile water of injectable grade (batch no. 1901341 obtained from Galenica Senese).

Evaluation criteria:

The test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95% control limits).
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data (95% control limits)

Statistics:

For the statistical analysis, a modified χ2 test was used to compare the number of cells with
micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to explanation for the third experiment, where dose levels spaced by a narrow interval were used, which ranged from the higher and lower doses tested in the previous experiments. (+ S9, highest dose: 100 µg/mL, -S9, highest dose 75 µg/mL).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no effect
- Data on osmolality: no effect
- Possibility of evaporation from medium:
- Water solubility: low (see preliminary study)
- Precipitation and time of the determination: no precipitation or opacity of the medium visible by eye was noticed at the beginning or by the end of treatment at any dose level.
- Definition of acceptable cells for analysis:
> The micronucleus diameter was less than 1/3 of the nucleus diameter.
> The micronucleus diameter was greater than 1/16 of the nucleus diameter.
> No overlapping with the nucleus was observed.
> Micronuclei were non-refractile and had the same staining intensity as the main nuclei.

PRELIMINARY STUDY:
This study was performed due to the low solubility of the test item in aqueous solvents. It was performed using DMSO, acetone and ethanol, with ethanol proved to be the best solvent and a clear solution was obtained at the concentration of 112 mg/mL corresponding to 100 mg/mL as active ingredient.

COMPARISON WITH HISTORICAL CONTROL DATA:
Following treatment with the test item, all incidences of micronucleated cells were within the distribution of historical negative control (95% confidence limits), no statistically significant increase over the concurrent solvent control value was observed at any concentration tested nor dose effect relationship was seen.

Incidence of micronucleated cells (%) after treatment with the test item in comparison to historical control data

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)	Cytotoxicity (%)	Presence of precipitation/ opacity
2	-	31	31	Untreated solvent 22.0 19.1 16.6	0.45 0.40 0.60 0.50 0.50	- - NS NS NS
				Linear trend                                               NS Historical control data (95% confidence limits)                0.00-1.00
3	-	3	32.5	Untreated solvent 14.8 9.88 6.60	0.30 0.30 0.55 0.20 0.15	- - NS NS NS
				Linear trend                                               NS Historical control data (95% confidence limits)                0.00-0.77
	+			Untreated solvent 22.2 14.8 9.88	0.30 0.20 0.40 0.15 0.50	-   NS NS NS
				Linear trend                                               NS Historical control data (95% confidence limits)                0.00-0.95

Conclusions:

On the basis of the results obtained, it is concluded that the test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. Three treatment conditions were performed. A short term treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of 32.5 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in ethanol. Based on the solubility of the test item, a Main Assay 1 was performed where treatments were as follows:

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)
1	-	3	32.5	250, 167, 111, 74.1, 49.4, 32.9, 21.9, 14.6 and 9.75
	+
	-	31	31	250, 167, 111, 74.1, 49.4, 32.9, 21.9, 14.6, 9.75 and 6.50

 

Since for all treatment series, a steep decline of cytotoxicity was observed and no concentration tested showed an adequate cytotoxicity to assess the frequency of micronucleated cells, a Main Assay 2 was performed where treatments were spaced by a narrower interval (1.15). Treatments were performed as follows:

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)
2	-	3	32.5	25.2, 21.9, 19.0, 16.5, 14.4, 12.5, 10.9, 9.46, 8.22 and 7.15
	+			33.3, 28.9, 25.2, 21.9, 19.0, 16.5, 14.4 and 12.5
	-	31	31	25.3, 22.0, 19.1, 16.6, 14.5, 12.6, 10.9, 9.51, 8.27 and 7.19

Possibly due to the properties of the test item, no reproducible results of cytotoxcity results were obtained with the short exposure both in the absence and presence of S9 metabolism. In agreement with the Sponsor, a further experiment (Main Assay 3), was performed by using dose levels spaced by a narrow interval and ranging from the higher and lower doses tested in the previous experiments. Treatments were as follows:

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)
3	-	3	32.5	75.0, 50.0, 33.3, 27.8, 22.2, 18.5, 14.8, 9.88 and 6.60
	+			100, 75.0, 50.0, 41.7, 33.3, 27.8, 22.2, 14.8, 9.88 and 6.60

 

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity. Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from a moderate to slight or no toxicity. Due to the steep decline of cytotoxicity observed after the short exposure both in the absence and presence of S9, the highest treatment level with no cytotoxicity obtained in Main Assay 3, was selected as the highest dose level for scoring. The next higher dose levels (18.5µg/mL and 27.8µg/mL respectively) could not be evaluated due to excessive cytotoxicity (very few or no cells). Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

 

Main Assay	S9	Treatment time (hours)	Harvest Time (hours)	Dose level (µg/mL)	Cytotoxicity (%)	Presence of precipitation/ opacity
2	-	31	31	0.00 22.0 19.1 16.6	- 41 8 5	- Np No No
3	-	3	32.5	0.00 14.8 9.88 6.60	- 8 10 9	- Np No No
	+			0.00 22.2 14.8 9.88	- 11 6 8	- Np No No

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in untreated and solvent control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine and the responses were compatible with those generated in our historical control database, indicating the correct functioning of the test system. The study was accepted as valid. Following treatment with the test item, all incidences of micronucleated cells were within the distribution of historical negative control (95% confidence limits), no statistically significant increase over the concurrent solvent control value was observed at any concentration tested nor dose effect relationship was seen.

It is concluded that the test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

 

 

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Jan. 6, 1995 to Jun. 27, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

CIBA-GEIGY Limited, Genetic Toxicology

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F10 medium supplemented with 10% pre-tested foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin in tissue culture.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary range finding test (with/without metbaolic activation) to assess cytotoxicty:
- concenration range: 0.195 to 400.00 µg/mL (highest dose due to solubility limitations)
- with metabolic activation:
400.00 µg/mL = 91.0% acute growth inhibiting effect
200.00 µg/mL = 76.00% acute growth inhibiting effect
- without metabolic activation:
down to 25.0 µg/mL = fully growth inhibiting
12.5 µg/mL = 99.3% acute growth inhibiting effect
6.25 µg/mL = 96.7% acute growth inhibiting effect

Accordingly, the following concentrations were selected for the original experiment:
Concentration range in the original test:
with S9 mix: 7.41, 22.22, 66.67 and 200.00 µg/ml
without S9 mix: 0.19, 0.56, 1.67 and 5.0 µg/ml

Due to the pronounced growth inhibitory response in the part with metabolic activation the concentration range was decreased in the confirmatory experiment:
Concentration range in the confirmatory test:
with S9 mix: 7.50, 15.0, 30.0 and 60.0 µg/ml
without S9 mix: 0.75, 1.50, 3:00 and 6.00 µg/ml

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with S9 mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours (with S9 mix), 21 hours (without S9 mix)
- Expression time (cells in growth medium): 7 to 8 days
- Selection time (if incubation with a selection agent): 7 to 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2.5-5.0 x 10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test substance will be considered to be mutagenic if:
- The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20;
-There is a significant dose-relationship as indicated by the linear trend analysis; and
-The effects described above are reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

200.0 µg/ml with metabolic activation and 5.0 µg/ml without metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test with and without metabolic activation 12 concentrations of the test substance were tested. The concentrations selected ranged from 0.195 to 400.00 µg/ml and separated by 2-fold intervals. In the part with metabolic activation, at the highest concentration of 400.0 µg/ml, an acute growth inhibiting effect of 91.0% could be seen, while the next lower concentration of 200.0 µg/ml inhibited 76.0%. Without metabolic activation treatment with the test substance proved fully growth inhibiting down to the concentration of 25.0 µg/ml. The next two lower concentrations of 12.5 and 6.25 µg/ml revealed an acute inhibition of growth of 99.3 and 96.7%, respectively. Accordingly, four concentrations were selected for the original experiment ranging from 7.41 to 200.0 µg/ml and from 0.19 to 5.0 µg/ml in the presence and absence of metabolic activation, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mutagenicity test with metabolic activation (Table 1):

The original experiment was performed at the following concentrations: 7.41, 22.22, 66.67 and 200.00 µg/ml. The highest concentration proved toxic after treatment. The mean growth inhibiting values found at the next lower concentration after treatment and expression were 98.9%* and 57.4%* respectively. In the confirmatory experiment the concentrations applied were 7.50, 15.0, 30.0 and 60.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 99.7%*. The mean growth inhibitory effect after the expression period was 65.5%*. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

 

Mutagenicity test without metabolic activation (Table 2):

The original experiment was performed at the following concentrations: 0.19, 0.56, 1.67 and 5.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 95.0%* and 31.9%* respectively. In the confirmatory experiment the concentrations applied were 0.75, 1.50, 3.00 and 6.00 µg/ml. The highest concentration proved toxic. The next lower concentration revealed a mean acute growth inhibitory effect of 70.5%*. The mean growth inhibition after the expression period was 11.6%*. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

*Mean of two independent cultures

 

Cytotoxicity determined after treatment (Table 3):

After treatment, the cytotoxicity was calculated from the cloning efficiency. Thereby, further dilution of the counted cell suspension/ concentration was performed (100 cells/9.6 cm² in growth medium; incubation at 37°C). The viability at the end of the treatment is reflected by the number of colonies which developed within 7-8 days (survival values).

 

Cytotoxicity determined after expression (Table 4):

At the end of the expression period, the viability was estimated from the cloning efficiency. The remaining cell suspensions from the various expression cultures were further diluted (100 cells/9.6 cm² in growth medium; incubation at 37°C). The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).

 

 

Table 1: With metabolic activation

Treatment	Mean mutant frequency (x 10E-6)	Mean mutant factor	Significance (P)
Original Experiment
Vehicle control	13.17	-	-
Positive control	172.14	13.07	P<0.001
TS, 200.00 µg/ml	*	*	*
TS, 66.67 µg/ml	19.60	1.49	0.001<p<0.002
TS, 22.22 µg/ml	14.35	1.09	Ns
TS, 7.41 µg/ml	12.46	0.95	Ns
Confirmatory Experiment
Vehicle control	7.26	-	-
Positive control	115.86	15.96	P<0.001
TS, 60.00 µg/ml	12.11	1.67	0.02<p<0.05
TS, 30.00 µg/ml	9.92	1.37	Ns
TS, 15.00 µg/ml	7.35	1.01	Ns
TS, 7.50 µg/ml	7.88	1.09	Ns

 

Table 2: Without metabolic activation

Treatment	Mean mutant frequency (x 10E-6)	Mean mutant factor	Significance (P)
Original Experiment
Vehicle control	13.78	-	-
Positive control	2203.81	159.90	P<0.001
TS, 5.00 µg/ml	19.56	1.42	P<0.001
TS, 1.67 µg/ml	13.52	0.98	Ns
TS, 0.56 µg/ml	13.35	0.97	Ns
TS, 0.18 µg/ml	11.57	0.84	Ns
Confirmatory Experiment
Vehicle control	7.48	-	-
Positive control	1147.38	153.46	P<0.001
TS, 6.00 µg/ml	*	*	*
TS, 3.00 µg/ml	5.78	0.77	Ns
TS, 1.50 µg/ml	2.51	0.34	Ns
TS, 0.75 µg/ml	5.35	0.72	Ns

TS: test substance; T: toxic; Ns: Not significant; *: no data

 

 

Table 3: Cytotoxicity determined after treatment

With metabolic activation
Treatment	Mean of survival clones	Number of viable cells (x10E6)	Acute cytotoxicity (% of control)
Original Experiment
Negative control	76.00	5.77
Negative control	76.50	5.98
Positive control, DMN 1 µl/ml	48.50	3.98	32.34
Positive control, DMN 1 µl/ml	55.83	4.54	22.75
TS 200.00 µg/mL	*	*	*
TS 200.00 µg/mL	*	*	*
TS 66.67 µg/mL	1.67	0.06	98.96
TS 66.67 µg/mL	1.50	0.06	98.91
TS 22.22 µg/mL	71.83	4.73	19.54
TS 22.22 µg/mL	72.67	4.49	23.52
TS 7.41 µg/mL	74.83	5.23	10.97
TS 7.41 µg/mL	71.33	4.44	24.40
Confirmatory Experiment
Negative control	73.00	5.31
Negative control	71.83	5.11
Positive control, DMN 1 µl/ml	49.17	2.89	44.59
Positive control, DMN 1 µl/ml	51.67	2.99	42.64
TS 60.00 µg/mL	0.67	0.02	99.70
TS 60.00 µg/mL	0.67	0.02	99.67
TS 30.00 µg/mL	21.00	0.79	84.79
TS 30.00 µg/mL	23.00	0.93	82.13
TS 15.00 µg/mL	67.00	2.93	43.67
TS 15.00 µg/mL	69.00	3.17	39.21
TS   7.50 µg/mL	68.17	3.28	37.01
TS   7.50 µg/mL	70.83	3.49	32.98
Without metabolic activation
Treatment	Mean of survival clones	Number of viable cells (x10E6)	Acute cytotoxicity (% of control)
Original Experiment
Negative control	78.17	7.69
Negative control	79.00	7.83
Positive control, EMS 0.3 µl/ml	28.17	3.81	50.92
Positive control, EMS 0.3 µl/ml	30.33	4.12	46.86
TS 5.00 µg/mL	24.67	0.37	95.18
TS 5.00 µg/mL	28.83	0.40	94.80
TS 1.67 µg/mL	82.17	5.14	33.78
TS 1.67 µg/mL	83.50	6.32	18.56
TS 0.56 µg/mL	83.00	7.94	nTx
TS 0.56 µg/mL	80.33	6.97	10.13
TS 0.18 µg/mL	80.33	6.37	17.98
TS 0.18 µg/mL	81.17	7.65	1.37
Confirmatory Experiment
Negative control	73.17	6.74
Negative control	71.83	6.92
Positive control, EMS 0.3 µl/ml	37.50	3.03	55.62
Positive control, EMS 0.3 µl/ml	37.50	3.29	51.91
TS 6.00 µg/mL	*	*	*
TS 6.00 µg/mL	*	*	*
TS 3.00 µg/mL	33.67	1.67	75.52
TS 3.00 µg/mL	42.33	2.36	65.48
TS 1.50 µg/mL	71.00	4.61	32.46
TS 1.50 µg/mL	74.00	4.94	27.67
TS 0.75 µg/mL	75.33	5.46	20.06
TS 0.75 µg/mL	71.67	5.32	22.10

*: No data

nTx: not toxic

Ns: not significant

 

Table 4: Cytotoxicity determined after expression

With metabolic activation
Treatment	Mean of viability clones	Number of viable cells (x10E6)	Cytotoxicity (% of control)
Original Experiment
Negative control	71.17	10.55
Negative control	69.33	10.84
Positive control, DMN 1 µl/ml	45.83	4.65	56.57
Positive control, DMN 1 µl/ml	44.50	5.04	52.90
TS 200.00 µg/mL	*	*	*
TS 200.00 µg/mL	*	*	*
TS 66.67 µg/mL	45.83	4.43	58.59
TS 66.67 µg/mL	49.83	4.69	56.16
TS 22.22 µg/mL	67.17	12.04	nTx
TS 22.22 µg/mL	68.00	12.29	nTx
TS 7.41 µg/mL	78.33	12.83	nTx
TS 7.41 µg/mL	78.17	12.78	nTx
Confirmatory Experiment
Negative control	78.50	10.60
Negative control	83.33	11.35
Positive control, DMN 1 µl/ml	61.00	7.49	31.74
Positive control, DMN 1 µl/ml	59.83	7.70	29.82
TS 60.00 µg/mL	46.00	3.86	64.82
TS 60.00 µg/mL	43.83	3.71	66.17
TS 30.00 µg/mL	70.67	8.47	22.85
TS 30.00 µg/mL	75.50	9.21	16.12
TS 15.00 µg/mL	74.67	10.10	7.93
TS 15.00 µg/mL	75.00	10.28	6.36
TS   7.50 µg/mL	84.83	11.79	nTx
TS   7.50 µg/mL	84.83	12.15	nTx
Without metabolic activation
Treatment	Mean of viability clones	Number of viable cells (x10E6)	Cytotoxicity (% of control)
Original experiment
Negative control	68.17	8.53
Negative control	71.50	10.17
Positive control, EMS 0.3 µl/ml	29.67	3.78	59.57
Positive control, EMS 0.3 µl/ml	27.17	3.05	67.35
TS 5.00 µg/mL	49.33	6.56	29.80
TS 5.00 µg/mL	51.00	6.17	33.99
TS 1.67 µg/mL	67.00	9.49	nTx
TS 1.67 µg/mL	71.67	9.97	nTx
TS 0.56 µg/mL	70.67	8.31	11.14
TS 0.56 µg/mL	68.83	8.87	5.17
TS 0.18 µg/mL	73.33	9.09	2.75
TS 0.18 µg/mL	76.83	10.21	nTx
Confirmatory experiment
Negative control	80.33	14.69
Negative control	81.83	15.37
Positive control, EMS 0.3 µl/ml	47.50	8.18	45.55
Positive control, EMS 0.3 µl/ml	48.00	8.28	44.91
TS 6.00 µg/mL	*	*	*
TS 6.00 µg/mL	*	*	*
TS 3.00 µg/mL	70.67	12.53	16.66
TS 3.00 µg/mL	78.50	14.04	6.62
TS 1.50 µg/mL	82.67	14.41	4.14
TS 1.50 µg/mL	86.50	15.08	nTx
TS 0.75 µg/mL	86.33	14.47	3.75
TS 0.75 µg/mL	88.83	15.46	nTx

*: No data

nTx: not toxic

Ns: not significant

Conclusions:

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test substance and its metabolites did not show any mutagenic activity.

Executive summary:

The test substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was dissolved in ethanol. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment. In a preliminary cytotoxicity test the concentrations for the main test were determined at 200.0 µg/ml and 5.0 µg/ml, with and without metabolic activation, respectively. The original experiment with S9-mix was performed at the following concentrations: 7.41, 22.22, 66.67 and 200.00 µg/ml. The highest concentration proved toxic after treatment. The mean growth inhibiting values found at the next lower concentration after treatment and expression were 98.9% and 57.4%, respectively. In the confirmatory experiment the concentrations applied were 7.50, 15.0, 30.0 and 60.0 µg/ml. The original experiment without S9-mix was performed at the following concentrations: 0.19, 0.56, 1.67 and 5.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 95.0% and 31.9%, respectively. In the confirmatory experiment the concentrations applied were 0.75, 1.50, 3.00 and 6.00 µg/ml. The highest concentration proved toxic. In both experiments with and without S9-mix, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). Therefore, based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test article and its metabolites did not show any mutagenic activity in this forward mutation system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Jun. 4, 1986 to Aug. 19, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

but QAU statement included

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentration in the preliminary toxicity test: 0.08 to 5000 µg/0.1 ml
Concentration range in the main tests (with and without S9 mix): 1.2, 4.9, 19.5, 78.1 and 312.5 µg/0.1 ml

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see Table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 ± 1.5 °C

NUMBER OF REPLICATIONS: 3 plates per test
-Independent repeat experiment was conducted.

Evaluation criteria:

The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

Revertant colony numbers were calculated and arithmetic mean and standard deviation were calculated. No significance test was performed.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

78.1 µg/0.1 ml (- S9) and 312.5 µg/0.1 ml (-S9 and +S9)

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 concentration of 312.5 µg/0.1 ml the substance precipitated in soft agar.

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/0.1 ml. Thereafter, the concentration of 312.5 µg/0.1 ml was used as the highest in the mutagenicity test due to a growth-inhibiting effect.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A growth-inhibiting effect of the test substance was observed in the experiments without microsomal activation at the upper concentrations. In the experiments carried out with microsomal activation, this effect was less pronounced.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Main test: Number of back-mutant colonies per plate (arithmetic mean)

Dose (µg/0.1ml)	TA 98		TA 100		TA 102		TA 1535		TA 1537
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0	41	24	114	151	246	274	12	12	20	7
1.2	45	24	103	133	243	277	12	13	16	6
4.9	48	27	122	155	285	226	12	15	16	8
19.5	34	30	122	78	110	250	10	15	12	6
78.1	43	3	152	6	3	212	8	1	11	1
312.5	26	1	10	0	9	4	6	1	4	1
PC*	1105	757	657	1035	822	1234	363	1018	126	929

 

Repeat test: Number of back-mutant colonies per plate (arithmetic mean)

Dose (µg/0.1ml)	TA 98		TA 100		TA 102		TA 1535		TA 1537
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0	41	25	119	122	254	306	14	13	9	6
1.2	40	25	113	117	252	299	16	18	11	5
4.9	38	20	97	130	235	262	10	15	10	8
19.5	48	20	108	99	216	123	15	11	6	4
78.1	40	6	131	5	179	2	16	2	11	1
312.5	16	2	27	5	2	0	1	0	1	0
PC	653	597	889	1045	1439	1146	375	904	75	1176

* Positive control (high dose)

Conclusions:

Based on the presented results and under the conditions employed, the test article did not induce point mutations in presence or absence of a metabolic activation system and is therefore regarded as not mutagenic in the Ames test.

Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES following OECD guideline No. 471 was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test article was applied by the plate incorporation method at concentrations of 1.2, 4.9, 19.5, 78.1 and 312.5 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates and repeated once for confirmation. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. A growth-inhibiting effect of the test substance was observed in the experiments without microsomal activation at the upper concentrations. In the experiments carried out with microsomal activation, this effect was less pronounced. At the concentration of 312.5 µg/0.1 ml the substance precipitated in soft agar. In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the substance is considered as not mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No clastogenic effects were observed in an in-vivo micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Jun. 2, 1986 to Nov. 3, 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

other: random outbred

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 21-32 g (females) and 23-35 g (males)
- Housing: individual caging
- Diet: NAFAG No. 924, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 3-4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23°C
- Humidity (%): 52-78%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 20 ml/kg

Details on exposure:

The preparation was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5000 mg/kg dose group. The positive control group consisted of 8 female and 8 male animals. Treatment consisted of a single application. 16, 24 and 48h after application 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.

Duration of treatment / exposure:

Single dose administered orally.

Frequency of treatment:

Once

Post exposure period:

16, 24, 48 hours after treatment application.

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

8/sex/sampling time

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 64 mg/kg in 20 ml/kg 0.5% CMC

Tissues and cell types examined:

Bone marrow was harvested from the shafts of both femurs.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. The highest dose causing no deaths is used as the highest in the mutagenicity test. No deaths were registered at any of the three groups of four Chinese hamsters (two female and two male animals ) treated with the doses 200, 1000 and 5000 mg/kg respectively, within the observation period of three days. Thus, the dose of 5000 mg/kg was chosen as no death occurred at this concentration.

TREATMENT AND SAMPLING TIMES:
16, 24 and 48h after application, 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a glass slide and the preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

METHOD OF ANALYSIS:
The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes were selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. 1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Evaluation criteria:

Statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at any sampling time.

Statistics:

The significance of difference was assessed by Chi square-test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the preliminary tolerability test, no deaths were registered at any of the three groups of four Chinese hamsters (two female and two male animals ) treated with the doses 200, 1000 and 5000 mg/kg respectively, within the observation period of three days. Thus, the dose of 5000 mg/kg was chosen for the micronucleus test as no death occurred at this concentration.

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: No statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times.
By contrast, the positive control (cyclophosphamide, 64 mg/kg) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 2.66. In comparison with the negative control (0.11) this value is highly significant (p<0.05).

Table: The effect of the test substance on bone marrow cells of Chinese hamster (arithmetic mean per sex and group).

Sacrifice	Dose	Sex	PCE	NCE	Ratio of PCE/NCE	No. of PCE with MN	% of PCE with MN
16 h	Control	Female	583	417	1.40	2.0	0.20
		Male	514	486	1.06	0.8	0.08
	TS	Female	650	350	1.86	0.6	0.06
		Male	511	489	1.04	1.2	0.12
24 h	Control	Female	595	405	1.47	1.2	0.12
		Male	562	438	1.28	1.0	0.10
	TS	Female	515	485	1.06	2.0	0.20
		Male	508	492	1.03	0.8	0.08
48 h	Control	Female	564	436	1.29	1.8	0.18
		Male	561	439	1.28	1.6	0.16
	TS	Female	520	480	1.08	0.6	0.06
		Male	456	544	0.83	1.0	0.10
Positive control
24 h	Control	Female	595	405	1.47	1.2	0.12
		Male	562	438	1.28	1.0	0.10
	CPA	Female	514	484	1.06	23.6	2.36
		Male	459	541	0.85	29.0	2.96

TS: test substance; PCE: polychromatic erythrocytes; NCE: normochromatic erythrocytes; MN: micronuclei; CPA: cyclophosphamide

Conclusions:

There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg bw of the test substance as compared with the negative control animals. Under the conditions of this experiment, no evidence of mutagenic effects was obtained in chinese hamster treated with the test substance.

Executive summary:

A micronucleus test in hamster was performed to evaluate any mutagenic effect of the test substance on polychromatic erythrocytes in bone marrow cells in vivo. To that end, the animals were treated once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. Mutagenic effects present themselves in form of micronuclei in polychromatic erythrocytes in the bone marrow. These micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes which lag behind at anaphase stage during the mitotic process. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm. The increase in micronucleated polychromatic erythrocytes shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations. In this experiment, the bone marrow smears from animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.66% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.11%) treated with the vehicle (0.5% CMC) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test

The mutagenic potential of the test substance was investigated in an Ames following OECD guideline 471, performed on Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of a metabolic activation system (S9 mix). None of the tested concentrations (1.2 to 312.5 µg/plate) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control either with or without metabolic activation. Owing to a growth-inhibiting effect of the substance a reduction in the colony count was observed in the experiments without microsomal activation at the upper concentrations and to a lesser extent with microsomal activation. This result was confirmed in a second independent experiment. Therefore, based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

 

HPRT-Test

In an HPRT-Test according to OECD guideline 474 and in compliance with GLP, the test substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro. Two independent experiments were performed. Concentrations range was from 7.41 to 200 µg/ml in the experiments with S9-mix; without S9-mix concentrations from 0.19 to 6.0 µg/ml were used. The test substance was dissolved in ethanol. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. Growth inhibiting effects were observed at high doses. In both experiments with and without S9-mix, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). Therefore, based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test article and its metabolites did not show any mutagenic activity in this forward mutation system.

In vitro MNT

In an in vitro MNT test according to OECD guideline 487 the ability of the test item to induce micronuclei in human lymphocytes cultured in vitro, after treatment in the absence and presence of S9 metabolism was assessed. Three treatment conditions were performed. A short treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of 32.5 hours, corresponding to approximately two cell cycle lengths, was used. A long term(continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in ethanol. Based on the solubility of the test item, a Main Assay 1 was performed with the following dose levels: 250, 167, 111, 74.1, 49.4, 32.9, 21.9, 14.6 and 9.75 µ/mL were used for the short treatment with and without S9. 250, 167, 111, 74.1, 49.4, 32.9, 21.9, 14.6, 9.75 and 6.50 µg/mL were used for the long treatment. Since for all treatment series, a steep decline of cytotoxicity was observed and no concentration tested showed an adequate cytotoxicity to assess the frequency of micronucleated cells, a Main Assay 2 was performed where treatments were spaced by a narrower interval (1.15). The doses 25.3, 22.0, 19.1, 16.6, 14.5, 12.6, 10.9, 9.51, 8.27 and 7.19 µg/mL were used for the long treatment in the absence of S9. Possibly due to the properties of the test item, no reproducible results of cytotoxcity results were obtained with the short exposure both in the absence and presence of S9 metabolism. In agreement with the Sponsor, a further experiment (Main Assay 3), was performed by using dose levels spaced by a narrow interval and ranging from the higher and lower doses tested in the previous experiments. The doses 75.0, 50.0, 33.3, 27.8, 22.2, 18.5, 14.8, 9.88 and 6.60 µg/mL were used for the short treatment in the absence of S9 and the doses 100, 75.0, 50.0, 41.7, 33.3, 27.8, 22.2, 14.8, 9.88 and 6.60 µg/mL were used for the short treatment in the presence of S9.  The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity.  Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from a moderate to slight or no toxicity. Due to the steep decline of cytotoxicity observed after the short exposure both in the absence and presence of S9, the highest treatment level with no cytotoxicity obtained inMain Assay 3, was selected as the highest dose level for scoring. The next higher dose levels (18.5μg/mLand 27.8μg/mLrespectively) could not be evaluated due to excessive cytotoxicity (very few or no cells). One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in untreated and solvent control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls  cyclophosphamide and Colchicine and the responses were compatible with those generated in our historical control database, indicating the correct functioning of the test system. The study was accepted as valid. Following treatment with the test item, all incidences of micronucleated cells were within the distribution of historical negative control (95% confidence limits), no statistically significant increase over the concurrent solvent control value was observed at any concentration tested nor dose effect relationship was seen.

It is concluded that the test item does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

In vivo micronucleus test

A micronucleus test in hamster was performed to evaluate any mutagenic effect of the test substance on polychromatic erythrocytes in bone marrow cells in vivo. To that end, the animals were treated once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. No statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals were observed at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.66% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.11%) treated with the vehicle (0.5% CMC) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.