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)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance DL-N33 was not mutagenic in the strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 in the presence and absence of S9 metabolic activation. (Annex V; Method B14).

In vitro cytogenicity (chromosome aberration) study in mammalian cells: DL-N33 did not induce any induce structural and/or numerical chromosomal damage in human lymphocytes in the presence or absence S9 metabolic activation (OECD 473/GLP).

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in mouse lymphoma L5178Y TK +/- cells exposed to DL-N33 in the presence or absence of phenobarbital and beta-naphthoflavone-induced rat liver S9 metabolic activation (OECD 476/GLP).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: SNIF

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

All information in this endpoint has been provided by the ECHA using the 12-year rule, this is data not owned by the registrant. The reliability is assumed to be at level 2. Therefore the following reliability statement can be used: Study conducted in accordance with generally accepted scientific principles, possibly with incorect reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

B14

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: Salmonella typhimurium, strains TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

50-5000 µg/plate

Vehicle / solvent:

Dimethylsulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Species / strain:

other: Salmonella typhimurium, strains TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Negative with or without metabolic activation.

Conclusions:

In a reverse bacteria mutation study (Ames test), DL-N33, was non-mutagenic in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (Annex V; Method B14), strains of S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 were exposed to DL-N33 in DMSO at concentrations of 50 - 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix).

There was no evidence of induced mutant colonies over background in the S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 strains in the presence and absence of mammalian metabolic activation.

Reference 2

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

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

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

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.

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'.

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

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

other: SNIF

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: See Remark

Remarks:

All information in this endpoint has been provided by the ECHA using the 12-year rule, this is data not owned by the registrant. The reliability is assumed to be at level 2. Therefore the following reliability statement can be used: Study conducted in accordance with generally accepted scientific principles, possibly with incorrect reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

2.5, 12.5 and 25 µg/ml (Above 25 µg/ml a precipitate formed in culture medium)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Fixation time:22 hrs

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Not toxic (no significant decrease in mitotic index)

Conclusions:

In an in vitro cytogenicity (chromosome aberration study) in human lymphocytes, DL-N33 did not induce any induce structural and/or numerical chromosomal damage in the presence or absence of rat liver S9 metabolic activation.

Executive summary:

In an in vitro cytogenicity (chromosome aberration) supporting study in mammalian cells (Annex V; Method B10), human lymphocytes were exposed to DL-N33 in DMSO at concentrations of 2.5, 12.5 and 25 µg/ml in the absence and presence of rat liver S9 metabolic activation system.

There was no cytotoxicity, but the substance was tested up to precipitating concentrations There was no evidence of a statistically significant increase in chromosomal aberrations with or without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study follows a recognised testing guideline and was performed under GLP.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

other: mouse lymphoma L5178Y TK +/- cells

Details on mammalian cell type (if applicable):

Cell lines (clone 3.7.2C) were obtained from Philip Morris Research Laboratories GmbH. These cell lines are heterozygous at the thymidine kinase locus.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix was obtained from RCC Cytotest Cell Research GmbH (Rossdorf, Germany). The homogenate was prepared from the livers of 8-10 week old male Wistar rats, treated with phenobarbital (intraperitoneally) and Beta-naphthoflavone (orally).

Test concentrations with justification for top dose:

A preliminary cytotoxicity test found little toxicity of T-77, and it was known to have very low water solubility. The highest concentration used in the preliminary test (900 micrograms/L) showed black precipitation: therefore this was chosen as the maximum concentration for the main test in the presence and absence of S9-mix.

Test concentrations used in the presence and absence of S9-mix: 30, 100, 300 and 900 micrograms/ml (nominal).

Vehicle / solvent:

Dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Remarks:

RPMI-1640 medium containing 25mM HEPES and 2mM L-glutamine supplemented with 5% heat inactivated horse serum

Negative solvent / vehicle controls:

yes

Remarks:

DMSO in culture medium supplemented with 5% heat inactivated horse serum

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9-mix: Methyl methanesulfonate (MMS); with S9-mix: Cyclophosphamide monohydrate (CP).

Details on test system and experimental conditions:

For cell propagation and the expression period, cells were cultured in plastic flasks in R10 culture medium with the following conditions: 37 degrees C, 5% CO2 and 90% humidity. TK+/- heterozygote cells are known to spontaneously mutate in culture to the homozygous TK-/- genotype and so before the L5178Y TK+/- cells were used they were cultured for 24h in the presence of thymidine, hypoxanthine, methotrexate and glycine to remove these TK-/- mutant cells. After this cells were washed and cultured for an additional 72h in methotrexate free medium.

Test procedure:

- Treatment of target cells: Aliquots of cells (10E06) from a proliferating culture were incubated at 37 +/- 2 degrees C for 4h in R5 culture media with or without 0.5% DMSO and in the presence of different concentrations of the test material, T-77 or reference substance. For cultures with metabolic activation, 1 ml of S9-mix was added, whereas for cultures without metabolic activation, 1ml KCl (150 mM) was added. Cultures were incubated in the dark whilst gently shaken.

Expression of the mutant phenotype: After the exposure period, cells were washed twice with R5 media, centrifuged and resuspended in R10 medium and counted. The black particulate test material could not be completely separated from the cells and this caused the net incubation time to become longer than 4h. After counting, 2 dilutions were made: 8 cells/ml in R20 medium to evaluate post-treatment cytotoxicity/viability and 2E05 cells/ml in R10 media for expression of the mutant phenotype.

For the mutant phenotype expression, 6E06 cells per 30 ml R10 media were cultured for an additional 2 days in culture flasks. Cells were counted and the population was adjusted to 6E06 cells after 24h. To determine post-treatment cytotoxicity (survivor I), cells were plated onto two 96-well plates (per treatment group) at a density of 1.6 cells/well in R20 culture media. These cells were grown for an additional 6 days until colonies were detectable and plating efficiency, relative survival and relative growth were calculated.

- Selection of the mutant phenotype: At the end of the expression period (48h), homozygous TK-/- mutants were selected by plating cells onto four 96-well plates per treatment group at a density of 2E03 cells/well. These cells were cultured in selective medium (R20 supplemented with 3 microgram/l TFT) for 14-15 days. After this period, mutant colonies were scored and plating efficiency and mutant frequencies were calculated.

To determine cytotoxicity/viability after the expression period (survivor II), cells were plated onto two 96-well plates (per treatment group) at a density of 1.6 cells/well in non-selective R20 culture media. These cells were grown for an additional 6 days until colonies were detectable and plating efficiency and relative survival were calculated. Viability was used to determine mutation frequency.

Viability was also assessed by microscopically determined cell counts immediately after treatment and over a 2-day expression period using a Neubauer haemocytometer. Suspension growth and total relative growth were also calculated.

Single cultures were used for each control and test treatment; independent replicate experiments were performed to verify results. In the main test, poor suspension growth or high spontaneous mutation rates led to rejection and repetition of individual experiments.

Evaluation criteria:

The study is considered valid if the following criteria are fulfilled:

- cells display normal morphology.
- Suspension growth values of negative controls are between 8 and 32.
- Plating efficiency (viable) of negative contols (calculated from survivor II data) are <0.65 or >1.2.
- There are at least 4 analysable concentrations with mutant frequency data.
- Spontaneous mutant frequencies are within the historical range or in the range of 50-200 E06 (as proposed by Moore et al, 2003).
- When compared to the background level, a significant increase in mutant frequency is observed with at least one concentration of each positive control (with and without S9-mix).
- The colony size distribution of the methyl methanesulphonate (MMS) positive controls demonstrates an increase in both small and large colonies.
- A relevant increase in mutation frequency (MF) is assessed by the 'Global Evaluation Factor' - a relevant increase in MF is if the MF of the test item amounted to more than MF negative control + 125.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

small RCC reduction, 900 micrograms/ml

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

900 micrograms/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary range-finding study (results are found in table 1):

Cytotoxicity was determined by measuring relative cell counts (RCC) directly after treatment with T-77. In the presence and absence of metabolic activation, signs of cytotoxicity were apparent, but these did not appear to be dose dependent. In the absence of metabolic activation, the RCC of all T-77 dose groups were reduced (at the 900 micrograms/ml T-77 dose group a maximum reduction to 81.4% of the vehicle control (0.5% DMSO) was observed). The relative survival (RS) reveals that no clear cytotoxic potential (although a reduction in RS was observed in the 50 micrograms/ml T-77 dose group). In the presence of metabolic activation, the RCC was reduced in some T-77 dose groups (except for the 10 and 25 micrograms/ml T-77 dose groups where the RCC was increased) with a maximal reduction to 81.7% of the vehicle control in the 900 micrograms/ml T-77 dose group. The RS was reduced at 1, 25, 150 and 300 micrograms/ml T-77, but these do not demonstrate dose dependency.


Main test:

In the absence of the metabolic activation:
The calculated mean suspension growth (SG), mean relative total growth (RTG), mean plating efficiency (survivor II PE) and mean relative survival (survivor II RS) demonstrate that no dose-dependent cytotoxicity is observed (see tables 2 and 3). Cell viability was reduced in the vehicle control as demonstrated by the lower RTG, PE and RS when compared to the negative control. As expected the positive control (MMS) showed that the mean SG and RTG were the lowest. As assessed by the concept of the Global Evaluation Factor, no significant increases in mutation frequency (MF) were observed in any T-77 dose group (see table 6 below). The spontaneous MF of the negative and vehicles were within the historical range (50E06 to 200E06). The positive control (MMS) showed a significant increase in mean MF.

In the presence of metabolic activation:
T-77 dose-dependent reduction in SG and RTG when compared to the vehicle control was observed (see tables 4 and 5). These reductions were maximal at 900 micrograms/ml T-77 (with an SG of 9.05 and RTG of 0.57 compared to the vehicle control of 15.75 and 1.07 respectively). Reductions in mean plating efficiency (survivor II PE) and mean relative survival (survivor II RS) were also observed (except in the 300 micrograms/ml group) but were not considered to be dose-dependent. The vehicle control showed higher or similar cell viability to the negative control. As expected the positive control (CP) showed a reduction in mean SG, RTG, PE (survivor I and II) and RS (survivor I and II). As assessed by the concept of the Global Evaluation Factor, T-77 did not cause a significant increase in MF (see table 7). Although slight MF increases were observed in the 100 and 300 micrograms/ml T-77 dose groups; these were not considered significant as they were within the historical range of the negative and vehicle controls. The spontaneous MF of the negative and vehicle controls were within the historical range (50E06 to 200E06). As expected the positive control (CP) caused a significant increase in mean MF.

Remarks on result:

other: other: Preliminary range-finding test

Remarks:

Migrated from field 'Test system'.

Table 1: Preliminary range-finding study.

	T-77 concentration (micrograms/ml)	Cell Count (x 10^6)	Relative Cell Count (%)	Number of wells scored	Nunber of colonies	Plating Efficiency Survivor I	Relative Survival (%)
Without S9-mix	0	5.9	100.0	96	61	0.63	100.0
	1	5.8	98.3	96	70	0.82	129.5
	10	5.3	89.8	96	74	0.92	146.0
	25	5.3	89.8	96	72	0.87	137.4
	50	5.5	93.2	96	56	0.55	86.8
	150	5.4	91.5	96	73	0.89	141.6
	300	4.9	83.1	96	80	1.12	177.6
	900	4.8	81.4	96	62	0.65	102.9
With S9-mix	0	6.0	100.0	96	76	0.98	100.0
	1	5.4	90.0	96	74	0.92	93.9
	10	6.3	105.0	96	80	1.12	114.2
	25	6.3	105.0	96	72	0.87	88.4
	50	5.8	96.7	96	79	1.08	110.4
	150	5.0	83.3	96	70	0.82	83.3
	300	5.7	95.0	96	66	0.73	74.1
	900	4.9	81.7	96	76	0.98	100.0

Table 2: Main test, cell and colony counts (survivor) without S9-mix

Treatment (micrograms/ml)		Replicate	Cell counts (x10^6)			Survivor I Colonies		Survivor II Colonies
			0h	24h	48h	Plate 1	Plate 2	Plate 1	Plate 2
Negative control		1	6.9	20.0	15.2	89	86	69	64
		2	11.6	22.5	28.5	75	67	67	66
Vehicle control		1	9.0	12.0	15.0	77	77	58	47
		2	11.2	22.0	27.5	63	61	63	54
T-77	30	1	11.1	13.3	18.8	76	74	59	67
		2	9.0	18.5	22.5	64	69	67	78
	100	1	10.8	13.3	18.3	56	62	59	53
		2	8.7	19.2	27.5	58	62	62	59
	300	1	10.0	14.4	17.1	79	71	66	62
		2	10.0	21.5	21.0	51	66	66	65
	900	1	10.0	14.1	22.2	74	66	54	60
		2	10.0	19.6	21.4	58	57	60	72
MMS (Positive control)	10	1	8.2	13.4	14.5	74	74	61	56
		2	7.1	24.0	17.5	69	64	60	57

Table 3: Main test, cytotoxicity/viability without S9-mix

Treatment (micrograms/ml)		Calculations including cell counts			Survivor I		Survivor II
		SG	%SG	RTG	PE	RS	PE	RS
Negative control		13.13	100.0	1.00	1.18	100.0	0.74	100.0
Vehicle control		10.91	83.1	0.57	0.83	72.0	0.54	74.4
T-77	30	9.25	70.4	0.75	0.85	75.2	0.77	104.9
	100	10.71	81.6	0.64	0.61	56.1	0.59	79.3
	300	9.69	73.8	0.71	0.77	66.3	0.71	95.1
	900	10.17	77.4	0.71	0.69	60.9	0.65	87.5
MMS (Positive control)	10	8.53	64.9	0.51	0.83	74.2	0.59	79.7

Table 4: main test, cell and colony counts (survivor) with S9 -mix

Treatment (microgram/ml)		Replicate	Cell counts (x10^6)			Survivor I colonies		Survivor II colonies
			0h	24h	48h	Plate 1	Plate 2	Plate 1	Plate 2
Negative control		1	9.4	21.3	28.0	82	82	66	73
		2	8.5	15.5	29.0	62	64	65	63
Vehicle control		1	9.4	25.0	27.0	80	82	69	69
		2	7.2	18.0	25.5	74	79	61	67
T-77	30	1	9.2	19.5	30.5	82	82	60	61
		2	9.3	19.5	28.0	62	60	66	62
	100	1	10.0	21.0	25.5	70	60	67	65
		2	9.7	16.5	25.5	69	65	59	53
	300	1	9.8	17.0	24.5	52	52	77	74
		2	8.9	15.5	22.5	70	71	68	55
	900	1	10.0	15.0	25.5	59	68	74	71
		2	8.0	12.5	21.5	69	70	51	56
CP (Positive control)	2.5	1	9.5	15.5	17.0	74	66	45	45
		2	7.6	9.6	19.5	62	50	35	40

Table 5: Main test, cytotoxicity/viability with S9-mix

Treatment (micrograms/ml)		Calculations including cell counts			Survivor I		Survivor II
		SG	%SG	RTG	PE	RS	PE	RS
Negative control		14.53	100.0	1.00	0.94	100	0.75	100.0
Vehicle control		15.75	108.4	1.07	1.08	122.8	0.74	99.3
T-77	30	15.84	109.1	0.99	0.92	97.2	0.65	88.6
	100	13.28	91.4	0.78	0.73	85.4	0.64	85.0
	300	10.63	73.2	0.78	0.66	82.3	0.80	106.5
	900	9.05	62.3	0.57	0.74	88.4	0.69	91.7
CP (Positive control)	2.5	6.26	43.1	0.2	0.68	74.9	0.35	47.1

Table 6: Main test, mutant frequencies, without S9 -mix

Treatment (micrograms/ml)		Mutant frequencies (TFT resistant mutants / 10^6viable cells)
		Mean Total colonies	Mean large colonies	Mean small colonies	Sum of large and small colonies	Proportion of small colonies (%)
Negative control		133.7	21.5	109.3	130.8	83.5
Vehicle control		135.5	15.5	119.0	134.5	88.4
T-77	30	118.7	14.1	102.1	116.2	87.9
	100	130.1	19.1	108.4	127.5	85.0
	300	104.7	16.7	85.7	102.4	83.6
	900	119.6	21.9	95.1	117.0	81.2
MMS (Positive control)	10	558.7	34.1	504.1	538.2	93.6

Table 7: Main test, mutant frequencies, with S9 -mix

Treatment (micrograms/ml)		Mutant frequencies (TFT resistant mutants / 10^6viable cells)
		Mean Total colonies	Mean large colonies	Mean small colonies	Sum of large and small colonies	Proportion of small colonies (%)
Negative control		94.8	27.9	64.7	92.6	69.8
Vehicle control		108.3	27.5	77.6	105.1	73.8
T-77	30	98.2	30.2	65.6	95.8	68.4
	100	120.5	35.1	82.9	118	70.2
	300	111.7	25.2	82.6	107.8	76.6
	900	91.7	22.7	66.8	89.5	74.6
CP (Positive control)	2.5	876.4	59.6	776	835.6	92.8

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the conditions of this study, in the presence and absence of metabolic activation T-77 did not cause a dose-dependent increase in gene mutations in mouse lymphoma L5178Y/TK+/- cells. All T-77 treated cultures demonstrated presence of MFs, but as these were in the normal range for the negative controls and were considered to be spontaneous mutations.

Executive summary:

In a mammalian cell gene mutation assay (TK locus), mouse lymphoma L5178Y cells cultured in vitro were exposed to T-77 at concentrations of 30, 100, 300 and 900 micrograms/mL in the presence and absence of mammalian metabolic activation (rat liver derived S9-mix)

 

There was no evidence of T-77 mutagenicity.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

There is one gene mutation study (Bacterial Reverse Mutation Assay/Ames test) available.

In a reverse gene mutation assay in bacteria (Annex V; Method B14), strains of S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 were exposed to DL-N33 in DMSO at concentrations of 50 - 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix).There was no evidence of induced mutant colonies over background in the S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 strains in the presence and absence of mammalian metabolic activation.

In vitro cytogenicity (chromosome aberration) study in mammalian cells:

There are 2 in vitro cytogenicity (chromosome aberration) studies available. The key study was guideline/GLP, had positive controls and extensive details.

In an in vitro cytogenicity (chromosome aberration) key study in mammalian cells (OECD 473/GLP), human lymphocytes were exposed to DL-N33 in DMSO at concentrations of 0-270 μg/mL (4 hrs) and 11.25-180 μg/mL (24 hrs) in the absence of S9 metabolic activation system and 0-360 μg/mL (4 hrs) and 22.5-270 (4 hrs) in the presence of S9 metabolic activation system.Cytotoxicity was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations. Positive controls induced the appropriate response. There was no evidence of a statistically significant increase in chromosomal aberrations with or without metabolic activation.

In an in vitro cytogenicity (chromosome aberration) supporting study in mammalian cells (Annex V; Method B10), human lymphocytes were exposed to DL-N33 in DMSO at concentrations of 2.5, 12.5 and 25 µg/ml in the absence and presence of rat liver S9 metabolic activation system.There was no cytotoxicity, but the substance was tested up to precipitating concentrations There was no evidence of a statistically significant increase in chromosomal aberrations with or without metabolic activation.

 

Gene mutation (mammalian cell gene mutation assay):

There is one gene mutation (mammalian cell gene mutation assay; MLA) available.

In an in vitro gene mutation study (MLA) in mammalian cells (OECD 476/GLP), mouse lymphoma L5178Y TK +/- cells were exposed to DL-N33 in DMSO at concentrations of 30-900µg/mL for 4 hrs with and without phenobarbital and beta-naphthoflavone-induced rat liver S9 metabolic activation. DL-N33 was tested up to the limits of cytotoxicity in the absence of metabolic acitvation and cytotoxicity was noted at 900µg/mL in the presence of metabolic acitvation. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

All studies are acceptable for use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance DL-N33 (CAS No. 104815-18-1) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.