Ferrate(1-), bis[4-[2-[5-chloro-2-(hydroxy-.kappa.O)phenyl]diazenyl-.kappa.N1]-3-(hydroxy-.kappa.O)-N-phenyl-2-naphthalenecarboxamidato(2-)]-, ammonium (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2011 - August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD guidline 473

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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:

-

EC Number:

403-590-1

EC Name:

-

Cas Number:

104815-18-1

Molecular formula:

C46 H32 Cl2 Fe N7 O6

IUPAC Name:

iron(3+) ammonium bis(1-[2-(5-chloro-2-oxidophenyl)diazen-1-yl]-3-(phenylcarbamoyl)naphthalen-2-olate)

Details on test material:

Sponsor's identification: DL-N33
Description: Black powder
Purity: 95%
Batch number: D33WC001
Date received: 28 March 2011
Expiry date: Not supplied
Storage conditions: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The purity of the test item was initially supplied as 85% and was accounted for in the formulations of the preliminary toxicity test. Subsequently the purity value was amended by the Sponsor to 95% and this value was used for the formulations of Experiment 1 and Experiment 2. The change in purity value resulted in the test item being tested above the maximum recommended dose level (5000µg/ml) in the preliminary toxicity test and the concentrations using both purity values have been recorded in the data.

Dose µg/ml (85%): 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000

Dose µg/ml (95%): 0, 21.6, 43.2, 86.4, 172.7, 345.4, 691, 1382, 2763, 5527

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

CELL CULTURE
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin Band 10% foetal bovine serum (FBS), at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

CULTURE CONDITIONS
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 ml MEM, 10% (FBS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

DURATION
- Preincubation period:
After approximately 48 hours incubation at 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 ml of the appropriate solution of vehicle control or test item was added to each culture.
After 4 hours at 37°C, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37°C in 5% CO2 in humidified air.

- Exposure duration:
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation.
Experiment 1
i) 4-hour exposure to the test item without 59-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 22.5 to 270 ~g/ml using a purity correction of 95%.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 22.5 to 360 ~g/ml using a purity correction of 95%.
Experiment 2
i) 24-hour continuous exposure to the test item without 59-mix prior to cell harvest. The dose range of test item used was 11 .25 to 180 IJg/ml using a purity correction of 95%.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 22.5 to 270 ~g/ml using a purity correction of 95%.

- Fixation time:
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 J.jg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCI. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCI cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4°C for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads
The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.
Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Statistics:

Statistical Analysis
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

mammalian cell line, other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/ml using a purity correction of 85% The maximum dose was based on the maximum recommended dose level. Subsequently the purity value was amended by the Sponsor to 95% which when applied to the dose levels of the Preliminary Toxicity Test gave a dose range of 21.6 to 5527 µg/ml, exceeding the maximum recommended dose level.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 625 (691)* µg/ml, in all three exposure groups. Both the blood cultures and parallel blood-free cultures were darker at all dose levels at the end of exposure due to the effects of the test item.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 312.5 (345.4)* µg/ml in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 156.25 (172.7)* µg/ml. Precipitate of the test item was observed on the slides at and above 1250 (1382)* µg/ml in all three exposure groups. The mitotic index data are presented in Table 1. The test item induced evidence of toxicity in all three of the exposure groups.

The selection of the maximum dose level for the main experiments was based on toxicity and was 270 µg/ml a.nd 360 µg/ml respectively for the 4(20)-hour exposure groups in the absence and presence of S9 in Experiment 1. In Experiment 2, 180 µg/ml was selected as the maximum dose for the continuous exposure group and 270 µg/ml for the 4(20)-hour exposure group with S9.

_____________________

* = figures in parenthesis show actual dose levels with a purity allowance of 95%

Experiment 1

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of DL-N33 (µg/ml)
4(20)-hour without S9	0*	22.5	45	90*	135*	180*	270	MMC 0.4*
4(20)-hour with S9 (2%)	0*	22.5	45*	90*	180*	270	360	CP 4.5*
* = Dose levels selected for metaphase analysis

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 270 µg/ml in both the presence and absence of metabolic activation (S9). A precipitate of the test item was observed at the end of exposure, at and above 45 µg/ml in both the 4 (20)-hour exposure groups and the cultures were darker at all dose levels due to the effects of the test item.

The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 52% and 70% mitotic inhibition was achieved at 180 µg/ml and 270 µg/ml respectively in the absence of S9. In the presence of S9 58% mitotic inhibition was achieved at 180 µg/ml.

The maximum dose level selected for metaphase analysis was based on toxicity and was 180 µg/ml for both the 4(20)-hour exposure groups.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Experiment 2

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of DL-N33 (µg/ml)
4(20)-hour without S9	0*	11.25*	22.5*	45*	90	135	180	MMC 0.2*
4(20)-hour with S9 (2%)	0*	22.5	45*	90*	135*	180	270	CP 4.5*
* = Dose levels selected for metaphase analysis

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 180 µg/ml in the presence of S9. In the absence of S9 the maximum test item dose level with metaphases was 135 µg/ml although due to the obvious toxicity at this dose level the maximum dose level selected for mitotic index analysis was 90 µg/ml. A precipitate of the test item was observed at the end of exposure, at and above 45 µg/ml both the 4 (20)-hour exposure group in the presence of S9 and the 24-hour continuous exposure group. The dose levels with the test item were all darker at the end of exposure due to the effects of the test item.

The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 58% and 64% mitotic inhibition was achieved at 135 and 180 µg/ml respectively in the presence of S9. In the 24-hour exposure group in the absence of S9, 68% mitotic inhibition was achieved at 45 µg/ml.

The maximum dose level selected for metaphase analysis was the based on toxicity and was 135 µg/ml for the 4(20)-hour exposure group in the presence of S9 and was 45 µg/ml for the 24-hour continuous exposure group in the absence of S9.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

TABLES

Mitotic Index - Preliminary Toxicity Test

4-HOUR TREATMENT, 20-HOUR RECOVERY -S9

4-HOUR TREATMENT, 20-HOUR RECOVERY +S9

24-HOUR TREATMENT -S9

CONCENTRATION (µg/ml)	4(20)h WITHOUT S9		4(20)h WITH S9		24h WITHOUT S9
	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX	% OF CONTROL
0	5.65	100	4.65	100	4.75	100
19.53 (21.6)	-	-	-	-	6.25	132
39.06 (43.2)	4.2	74	4.9	105	3.95	83
78.13 (86.4)	4.4	78	5.1	110	1.75	37
156.25 (172.7)	3.15	56	3.2	69	0.3	6
312.5 (345.4)	1.1	19	0.15	3	NM	0
625 (691)	NM P	0	NM P	0	NM P	0
1250 (1382)	NM P*	0	NM P*	0	NM P*	0
2500 (2763)	NM P*	0	NM P*	0	NM P*	0
5000 (5527)	NM P*	0	NM P*	0	NM P*	0
Figures in parenthesis show actual dose levels with a purity allowance of 95% - = Not assessed for mitotic index NM = No metaphases suitable for scoring P = Precipitate observed at end of exposure period in blood-free cultures P* = Precipitate observed at end of exposure period in blood-free cultures and also on the slides

Mitotic Index - Experiment 1

DOSE LEVEL (µg/ml)	4 HOURS TREATMENT WITHOUT S9				4 HOURS TREATMENT WITH S9
	A	B	MEAN	% OF CONTROL	A	B	MEAN	% OF CONTROL
0	7.9	6.95	7.43	100	6.9	5.6	6.25	100
22.5	-	-	-	-	-	-	-	-
45	- P	- P	-	-	6.58 P	7.15 P	7	112
90	7.00 P	5.50 P	6.25	84	5.80 P	5.55 P	5.68	91
135	4.35 P	4.00 P	4.18	56	NA	NA	NA	NA
180	3.20 P	3.95 P	3.58	48	2.85 P	2.45 P	2.65	42
270	1.75 P	2.65 P	2.2	30	0.60 P	0.80 P	0.7	11
360	NA	NA	NA	NA	NM	NM	NM	NM
MMC 0.4	2.4	3.25	2.86	38	NA	NA	NA	NA
CP 4.5	NA	NA	NA	NA	2.15	1.9	2.03	32
MMC = Mitomycin C CP = Cyclophosphamide P = Precipitate NA = Not applicable - = Not assessed for mitotic index NM = No metaphases suitable for scoring

Mitotic Index - Experiment 2

DOSE LEVEL (µg/ml)	4 HOURS TREATMENT WITHOUT S9				4 HOURS TREATMENT WITH S9
	A	B	MEAN	% OF CONTROL	A	B	MEAN	% OF CONTROL
0	3.75	3.15	3.45	100	5.15	5.55	5.35	100
11.25	4.05	4.35	4.2	122	NA	NA	NA	NA
22.5	3	3.1	3.05	88	-	-	-	-
45	1.35 P	0.85 P	1.1	32	4.80 P	5.05 P	4.93	92
90	0.25 P	0.20 P	0.23	7	4.85 P	4.50 P	4.68	87
135	NM P	NM P	NM	NM	1.90 P	2.60 P	2.25	42
180	NM P	NM P	NM	NM	2.75 P	1.10 P	1.93	36
270	NA	NA	NA	NA	NM P	NM P	NM	NM
MMC 0.2	1.7	0.9	1.3	38	NA	NA	NA	NA
CP 4.5	NA	NA	NA	NA	2.3	1.7	2	37
CP = Cyclophosphamide MMC = Mitomycin C P = Precipitate NA = Not applicable = Not assessed for mitotic index NM = No metaphases suitable for scoring

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce a statistically significant increase in the frequency of cells
with chromosome aberrations in either the absence or presence of a liver enzyme
metabolising system in either of two separate experiments. The test item was therefore
considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method was designed to be compatible with that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration of DL-N33 (µg/ml)
4(20)-hour without S9	22.5	45	90	135	180	270
4(20)-hour with S9 (2%)	22.5	45	90	180	270	360
24-hour without S9	11.25	22.5	45	90	135	180
4(20)-hour with S9 (1%)	22.5	45	90	135	180	270

Results

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that induced approximately 50% mitotic inhibition or greater.

Conclusion

The test item was considered to be non-clastogenic to human lymphocytes in vitro.