1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 July- 22 November, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29th July, 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

EC Number:

290-904-3

EC Name:

Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

Cas Number:

90268-98-7

Molecular formula:

Molecular formula is not available

IUPAC Name:

Reaction product of aniline, 4-nitrobenzenamine, p-phenylenediamine and p-toluidine with sodium polysulfide

Test material form:

solid: particulate/powder

Details on test material:

Test item: Yellow 22
Appearance: ocher clay, solid
CAS No. 90268-98-7
EC No. 290-904-3
Storage: room temperature

Specific details on test material used for the study:

Date of production: 22.05.2015
Expiration date: 22.05.2020

Method

Target gene:

Chromatid and chromosome type aberrations in metaphase cells

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79: Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with
L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.

Cytokinesis block (if used):

Cell cultures were treated with colchicine (0.2 µg/mL) 2.5 hours prior to harvesting

Metabolic activation:

with and without

Metabolic activation system:

liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 40.3 and 33.8 mg/mL.

Test concentrations with justification for top dose:

Experiment A (3/20h): 15.6,1 31.3, 62.5, 125 and 1802 μg/mL test item with and without S9 mix
Experiment B (20/20h and 20/28h): 3.9, 1 7.8, 15.6, 31.3 and 452 μg/mL test item without S9 mix
Experiment B (3/28h): 15.6, 1 31.3, 62.5, 125 and 180 μg/mL test item with S9 mix

Vehicle / solvent:

Dimethyl sulfoxide DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: DME (Dulbecco’s Modified Eagle’s) medium

Details on test system and experimental conditions:

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations.
Mammalian Microsomal Fraction S9 Mix

An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 40.3 and 33.8 mg/mL.

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.

The S9 Mix (with Rat Liver S9)

The complete S9 Mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL
HEPES* 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**= β-Nicotinamide Adenine Dinucleotide Phosphate

Before adding to the culture medium the S9 mix was kept in an ice bath.

Rationale for test conditions:

Acceptability of the Assay

The chromosome aberration assay is considered acceptable because it meets the following criteria:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data,
– concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
– cell proliferation in the solvent control is adequate,
– adequate number of cells and concentrations are analyzable,
– all requested experimental conditions were tested unless one resulted in a positive result
– the criteria for the selection of top concentration are fulfilled.

Evaluation criteria:

Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.


Interpretation of Results

Providing that all acceptability criteria are fulfilled, the test item is considered to be clearly positive if, in any of the experimental conditions examined:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and Dose Selection

A homogeneous suspension of the test item was obtained in DMSO up to a concentration of 100 mg/mL. There was no precipitation in the medium at any concentration tested.
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix). Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Detailed results of the cytotoxicity assay with the test item are presented in Table 2A - 2C.
Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:

Experiment A with 3/20 h treatment/sampling time
without: 15.6,1 31.3, 62.5, 125 and 1802 *g/mL test item
with S9 mix: 15.6,1 31.3, 62.5, 125 and 180 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 3.9, 1 7.8, 15.6, 31.3 and 452 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 3.9, 1 7.8, 15.6, 31.3 and 452 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 15.6, 1 31.3, 62.5, 125 and 180 *g/mL test item
1These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next higher concentration and sufficient number of concentrations.
2These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations.

All concentrations were run in duplicates (incl. negative and positive controls) and 300 (150 per culture) well-spread metaphases were assessed.

Chromosome Aberration Assay

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item (Tables 13 and 14).

In both experiments, clear cytotoxicity of about 50% was observed after test item treatment in the absence and presence of metabolic activation.

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation
In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values (5-6 aberrant cells excluding
gaps/150 cells) were slightly above the 95% control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells). However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant.

No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.

The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 *L/mL) and Cyclophosphamide (5 *g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data .Thus, the study is considered valid.

Remarks on result:

other: In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Remarks:

test item is considered as being non-clastogenic in this system

Any other information on results incl. tables

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

3-hour treatment without and with S9 mix / 20-hour sampling time

 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	1850000	1900000	1918750	-	-	-
	-	B	–	2050000	1950000
	-	C	–	2000000	1800000
	-	D	–	1850000	1950000
Solvent control (DMSO)	-	A	–	6700000	7000000	6862500	4943750	100,00	0,00
	-	B	–	6850000	6900000
test item	31.3	A	–	6800000	6800000	6800000	4881250	98,74	1,26
	62.5	A	–	5400000	5150000	5275000	3356250	67,89	32,11
	125	A	–	4300000	4400000	4350000	2431250	49,18	50,82
	250	A	–	3150000	3000000	3075000	1156250	23,39	76,61
	500	A	–	2500000	2450000	2475000	556250	11,25	88,75
	EMS 1 µL/mL	A	–	4600000	4600000	4600000	2681250	54,24	45,76
Solvent control (DMSO)	-	A	+	6600000	6850000	6700000	4781250	100,00	0,00
	-	B	+	6650000	6700000
test item	31.3	A	+	6600000	6600000	6600000	4681250	97,91	2,09
	62.5	A	+	5800000	6100000	5950000	4031250	84,31	15,69
	125	A	+	4400000	4200000	4300000	2381250	49,80	50,20
	250	A	+	3600000	3700000	3650000	1731250	36,21	63,79
	500	A	+	3250000	3250000	3250000	1331250	27,84	72,16
	Cycl. 5µg/mL	A	+	4350000	4500000	4425000	2506250	52,42	47,58

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

DME: (Dulbecco’s Modified Eagle’s)medium

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

20-hour treatment without S9 mix / 20-hour sampling time

 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	1850000	1900000	1918750	-	-	-
	-	B	–	2050000	1950000
	-	C	–	2000000	1800000
	-	D	–	1850000	1950000
Solvent control (DMSO)	-	A	–	5400000	5500000	5487500	3568750	100,00	0,00
	-	B	–	5600000	5450000
test item	7.8	A	–	5500000	5450000	5475000	3556250	99,65	0,35
	15.7			4250000	4500000	4375000	2456250	68,83	31,17
	31.3	A	–	3600000	3700000	3650000	1731250	48,51	51,49
	62.5	A	–	3000000	3000000	3000000	1081250	30,30	69,70
	125	A	–	1800000	1700000	1750000	-168750*	-4,73**	104,73***
	250	A	–	1250000	1150000	1200000	-718750*	-20,14**	120,14***
	500	A	–	900000	1000000	950000	-968750*	-27,15**	127,15***
	EMS 1 µL/mL	A	–	3600000	3550000	3575000	1656250*	46,41**	53,59***

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

DME: (Dulbecco’s Modified Eagle’s)medium

EMS: Ethyl methanesulfonate (EMS)

*: cell number decrease,

**: zero RICC value,

***:100% cytotoxicity

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time

 

Test group	Concentration (µg/mL)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	1850000	1900000	1918750	-	-	-
	-	B	–	2050000	1950000
	-	C	–	2000000	1800000
	-	D	–	1850000	1950000
Solvent control (DMSO)	-	A	–	8600000	8350000	8450000	6531250	100,00	0,00
	-	B	–	8500000	8350000
test item	7.8	A	–	8200000	8300000	8250000	6331250	96,94	3,06
	15.7	A	–	6950000	6750000	6850000	4931250	75,50	24,50
	31.3	A	–	5150000	5000000	5075000	3156250	48,33	51,67
	62.5	A	–	3850000	3600000	3725000	1806250	27,66	72,34
	125	A	–	1900000	1850000	1875000	-43750*	-0,67**	100,67***
	250	A	–	1300000	1300000	1300000	-618750*	-9,47**	109,47***
	500	A	–	700000	600000	650000	-1268750*	-19,43**	119,43***
	EMS 1 µL/mL	A	–	4900000	5050000	4975000	3056250*	46,79**	53,21***
Solvent control (DMSO)	-	A	+	8800000	8950000	8762500	6843750	100,00	0,00
	-	B	+	8600000	8700000
test item	31.3	A	+	8800000	8550000	8675000	6756250	98,72	1,28
	62.5	A	+	7600000	7500000	7550000	5631250	82,28	17,72
	125	A	+	5200000	5300000	5250000	3331250	48,68	51,32
	250	A	+	4400000	4600000	4500000	2581250	37,72	62,28
	500	A	+	3900000	4000000	3950000	2031250	29,68	70,32
	Cycl. 5µg/mL	A	+	5150000	4950000	5050000	3131250	45,75	54,25

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT A

 

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Negative (Solvent) control	-	3 h	20 h	7	3
test item
31.3 µg/mL	-	3 h	20 h	12	5
62.5 µg/mL	-	3 h	20 h	12	5
125 µg/mL	-	3 h	20 h	11	6
Pos. Control (Ethyl methanesulphonate)	-	3 h	20 h	41**	31**
Negative (Solvent) control	+	3 h	20 h	8	4
test item
31.3 µg/mL	+	3 h	20 h	10	5
62.5 µg/mL	+	3 h	20 h	12	5
125 µg/mL	+	3 h	20 h	14	6
180 µg/mL	+	3 h	20 h	10	5
Pos. Control (Cyclophosphamide)	+	3 h	20 h	45**	41**

Positive control (-S9): Ethyl methanesulphonate (1.0L/mL)

Positive control (+S9): Cyclophosphamide (5.0g/mL)

**= p < 0.01 to the concurrent negative control and to the historical control

 

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B

 

Concentration (µg/mL)		S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
					incl. gaps	excl. gaps
	Negative (Solvent) control	-	20 h	20 h	7	3
	test item
	7.8 µg/mL	-	20 h	20 h	8	3
	15.6 µg/mL	-	20 h	20 h	8	4
	31.3 µg/mL	-	20 h	20 h	8	4
	Pos. Control (Ethyl methanesulphonate)	-	20 h	20 h	47**	38**
	Negative (Solvent) control	-	20 h	28 h	7	3
	test item
	7.8 µg/mL	-	20 h	28 h	8	3
	15.6 µg/mL	-	20 h	20 h	7	3
	31.3 µg/mL	-	20 h	28 h	7	4
	Pos. Control (Ethyl methanesulphonate)	-	20 h	28 h	48**	37**

Positive control (-S9): Ethyl methanesulphonate (0.4L/mL)

**= p < 0.01 to the concurrent negative control and to the historical control

 

TABLE 9 continued

 

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B

 

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150cells
				incl. gaps	excl. gaps
Negative (Solvent) control	+	3 h	28 h	7	4
test item
31.3 µg/mL	+	3 h	28 h	8	3
62.5 µg/mL	+	3 h	28 h	10	5
125 µg/mL	+	3 h	28 h	10	3
180 µg/mL	+	3 h	28 h	11	5
Pos. Control (Cyclophosphamide)	+	3 h	28 h	48**	39**

Cyclophosphamide: 5.0g/mL

**= p < 0.01 to the concurrent negative control and to the historical control

 

 

APPENDIX IV

 

 

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT A

 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	3/20 h	0.0	0.0
test item
31.3 µg/mL	-	3/20 h	0.0	0.0
62.5 µg/mL	-	3/20 h	0.0	0.0
125 µg/mL	-	3/20 h	0.0	0.0
Pos. Control (Ethyl methanesulphonate)	-	3/20 h	0.0	0.0
Negative (Solvent) control	+	3/20 h	0.0	0.0
test item
31.3 µg/mL	+	3/20 h	0.0	0.0
62.5 µg/mL	+	3/20 h	0.0	0.0
125 µg/mL	+	3/20 h	0.0	0.0
180 µg/mL	+	3/20 h	0.0	0.0
Pos. Control (Cyclophosphamide)	+	3/20 h	0.0	0.0

Ethyl methanesulphonate: 1.0mL/mL

Cyclophosphamide: 5.0g/mL

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

 

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT B

 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	20/20 h	0.0	0.0
test item
7.8 µg/mL	-	20/20 h	0.0	0.0
15.6 µg/mL	-	20/20 h	0.0	0.0
31.3 µg/mL	-	20/20 h	0.0	0.0
Pos. Control	-	20/20 h	0.0	0.0
Negative (Solvent) control	-	20/28 h	0.0	0.0
test item
7.8 µg/mL	-	20/28 h	0.0	0.0
15.6 µg/mL	-	20/28 h	0.0	0.0
31.3 µg/mL	-	20/28 h	0.0	0.0
Pos. Control	-	20/28 h	0.0	0.0

Positive control (-S9):Ethyl methanesulphonate(0.4L/mL)

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

 

TABLE 11 Continued

 

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT B

 

Concentration (µg/mL)	S9 mix	Treatment/Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	+	3/28 h	0.0	0.0
test item
31.3 µg/mL	+	3/28 h	0.0	0.0
62.5 µg/mL	+	3/28 h	0.0	0.0
125 µg/mL	+	3/28 h	0.0	0.0
180 µg/mL	+	3/28 h	0.0	0.0
Pos. Control	+	3/28 h	0.0	0.0

Cyclophosphamide: 5.0g/mL

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

Applicant's summary and conclusion

Conclusions:

The test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Executive summary:

The test item suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473.

Experiment A with 3/20 h treatment/sampling time

without: 15.6,1 31.3, 62.5, 125 and 1802g/mL test item

with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the test item is considered as being non-clastogenic in this system.