Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD Guideline 471 (Bacterial Reverse Mutation Assay): The test item was considered to be non-mutagenic under the conditions of this test.

OECD Guideline 473 (chromosome aberration test): Pigment Red 81:4 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 metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

OECD Guideline 473 ( L5178Y TK +/- Mouse Lymphoma Assay): The test item did not induce any increase in mutant frequency at the TK +/- locus L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 07 January 2014; Experimental Completion Date: 03 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

Species / strain / cell type:

other: S. typhimurium strains TA1535, TA1537, TA98 and TA100. E. coli strain WP2uvrA

Details on mammalian cell type (if applicable):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

Test concentrations with justification for top dose:

Experiment 1 (Plate incorporation Method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (Pre-Incubation Method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: In solubility checks performed in–house, the test item was insoluble in sterile distilled water and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL but was soluble in dimethyl sulphoxide and dimethyl formamide at 50 mg/mL. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

The positive controls ENNG, 9AA and 4NQO were used in the series of plates without S9-mix. 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP) were used in the series of plates with S9-mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical analysis of data as determined by UKEMS.

Species / strain:

other: S. typhimurium strains TA1535, TA1537, TA98 and TA100. E. coli strain WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no toxicologically significant increases in the frequency of revertant colonies

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results for details

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In Experiment 1 (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the tester strains in both the absence and presence of S9-mix, initially from 1500 µg/plate (TA1537) and at 5000 µg/plate (remaining strains). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second experiment. In Experiment 2 (pre-incubation method) the test item induced a stronger toxic response with either a visible reduction in the growth of the bacterial background lawn and/or a substantial reduction in the frequency of revertant colonies noted to all of the tester strains, initially from 500 µg/plate (E.coli strain WP2uvrA, TA1537 with and without S9 and TA1535 in the absence of S9 mix only) and from 1500 µg/plate to the remaining strains. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. In Experiment 1 (plate incorporation method), an intense pink test item induced coloration was noted from 1500 µg/plate with an associated precipitate (powdery in appearance) observed at 5000 µg/plate. In Experiment 2, after the change in test methodology, an intense pink test item induced coloration was only noted at 1500 µg/plate and the associated precipitate was much thicker at 5000 µg/plate. None of these observations prevented the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Small, statistically significant increases in TA1537 revertant colony frequency were noted in the absence of S9 mix at 150 and 500 µg/plate. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 and 500 µg/plate were within the in house historical untreated/vehicle control range for the tester strain. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Pigment Red 81:4 was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 12 January 2018 Experimental completion date 27 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation test

Specific details on test material used for the study:

Identification: Pigment Red 81:4
Physical state/Appearance: Red powder
Batch: 64078
CAS Number: 85959-61-1
Purity: 97%
Expiry Date: 30 June 2018
Storage Conditions: Room temperature, in the dark
A correction for the purity of the test item was applied.

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
0, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 μg/ml
The molecular weight of the test item was 486.64, therefore the maximum dose level was 2000 μg/mL, the maximum recommended dose level. The purity of the test item was 97% and was therefore accounted for when formulating the dosing solutions

Main test:
4-hour treatment (-S9): 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32 μg/ml
4-hour treatment (+S9): 0, 0.5, 1, 2, 4, 8, 16, 32, 64 μg/ml
24-hour treatment (-S9): 0, 0.015, 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 μg/ml
the maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO
Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups

Positive control substance:

ethylmethanesulphonate

Remarks:

absence of metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1.5 μg/mL in 4 hour exposure group

Positive control substance:

cyclophosphamide

Remarks:

presence of metabolic activation

Details on test system and experimental conditions:

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO Acros Organics batch A0385951, purity 99.7%, expiry 01/06/20) exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBS6100V, purity treated as 100%, expiry 17.11.22) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0373263, purity 97%, Expiry 22.02.19) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. Due to the high levels of precipitate observed in the solubility test, the dose range used in the preliminary toxicity test was 1.95 to 500 μg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB*) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106^ cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (0.25 to 32 μg/mL for the 4-hour exposure group in the absence of metabolic activation, 0.5 to 64 μg/mL for the 4-hour exposure group in the presence of metabolic activation, and 0.015 to 2 μg/mL for the 24-hour exposure group in the absence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Evaluation criteria:

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups, with the greatest reduction observed in the 24-hour exposure group in the absence of metabolic activation, when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at and above 125 μg/mL in all three of the exposure groups at the end of the exposure period. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose level in the subsequent Mutagenicity Test was limited by test item-induced toxicity.

Mutagenicity Test
There was evidence of marked dose related toxicity following exposure to the test item in all of the three exposure groups, as indicated by the %RSG and RTG values. There was also evidence of modest reductions in viability (%V) in all three of the exposure groups, indicating that residual toxicity had occurred. Based on the %RSG and RTG values observed, optimum levels of toxicity were achieved in all three of the exposure groups. Acceptable levels of toxicity were seen with the positive control substances.
At the end of the exposure periods, no precipitate of the test item was observed in any of the three exposure groups.
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels (including the dose levels that achieved optimum levels of toxicity), in any of the three exposure groups.

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 1.95 to 500 μg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
1.95	39	47	1
3.91	32	33	1
7.81	31	22	1
15.63	24	26	0
31.25	16	20	0
62.5	3	16	0
125	1	3	0
250	0	1	0
500	0	0	0

Summary of Results

Concentration (µg/mL)			4-Hours-S9					Concentration (µg/mL)			4-Hours+S9
			%RSG	RTG	MF§						%RSG	RTG	MF§
0			100	1.00	133.93			0			100	1.00	146.31
0.25		Ø	80					0.5		Ø	104
0.5		Ø	60					1		Ø	95
1			48	0.50	147.44			2			74	0.73	163.05
2			34	0.39	135.69			4			46	0.48	126.16
4			27	0.30	134.18			8			31	0.34	127.14
8			25	0.27	134.52			16			27	0.29	120.23
16			23	0.21	137.44			32			21	0.20	101.25
32			16	0.13	127.21			64			16	0.14	139.22
MF threshold for a positive response = 259.93								MF threshold for a positive response = 272.31
Positive control								Positive control
EMS								CP
400			82	0.53	1096.63			1.5			77	0.51	924.98

Concentration (µg/mL)		24-Hours-S9
		%RSG	RTG	MF§
0		100	1.00	143.78
0.015	Ø	105
0.03		103	0.96	137.43
0.06		108	1.05	128.48
0.13		99	0.99	142.14
0.25		55	0.63	132.37
0.5		20	0.37	116.68
1		8	0.11	124.97
2	Ø	3
MF threshold for a positive response = 269.78
Positive control
EMS
150		61	0.44	1650.37

$ = Cell counts (x10^5 cells/mL). Set up on previous day to 2 x 10^5 cells/mL unless otherwise stated in parenthesis.

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

%V = Viability Day 2

§ or # = Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

A,B = Replicate cultures

CP = Cyclophosphamide

EMS = Ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/106 viable cells 2 days after exposure

Nv = Number of wells scored, viability plates

Yv = Number of wells without colonies, viability plates

Ym = Number of wells without colonies, mutation plates

Nm = Number of wells scored, mutation plates

Ø = Not plated, surplus to requirements

NP = Not plated for viability or 5-TFT resistance

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group	Concentration of Pigment Red 81:4 (µg/mL) plated for viability and mutant frequency
4-hour without S9	1, 2, 4, 8, 16, 32
4-hour with S9 (2%)	2, 4, 8, 16, 32, 64
24-hour without S9	0.03, 0.06, 0.13, 0.25, 0.5, 1

Results

The maximum dose levels in the Mutagenicity Test were limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 16 January 2018 Experimental completion date 21 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: Pigment Red 81:4
Physical state/Appearance: Red powder
Batch: 64078
Purity: 97%
Expiry Date: 30 June 2018
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (3%) of the test item.

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human whole blood

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 28 years Main Experiment: male, aged 28 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
All exposure groups: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 μg/mL
Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The molecular weight of the test item was 486.64, therefore the proposed maximum dose level was 2000 μg/mL, the maximum recommended dose level. However, marked precipitate was observed at and above 250 μg/mL in the solubility test and, therefore, this was adopted as the revised maximum dose level. The purity of the test item was 97% and was therefore accounted for when formulating the dosing solutions.


Main experiment:
4-hour exposure group (-S9): 0, 0.025, 0.5, 1, 2, 8, 16 μg/mL
4-hour exposure group (+S9): 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg/mL
24-hour exposure group (-S9): 0, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2 μg/mL
The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/mL for 4-hour exposure and 24-hour exposure

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2 μg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Test Item Preparation and Analysis
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

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 heparinized whole blood.

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 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. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
After 4 hours at approximately 37 ºC, 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 approximately 37 ºC in 5% CO2 in humidified air.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was 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 reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-Hour Exposure Without Metabolic Activation (S9)
As the exposure was continuous, the cultures were established at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
The Preliminary Toxicity Test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 0.98 to 250 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the Main Experiment.

Main Experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0.25, 0.5, 1, 2, 4, 8 and 16 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0.25, 0.5, 1, 2, 4, 8, and 16 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0.031, 0.063, 0.125, 0.25, 0.5, 1 and 2 μg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/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 re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl 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 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension 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 labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation criteria:

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Species / strain:

lymphocytes: human whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 μg/mL. The maximum dose was limited to 250 μg/mL due to the precipitate and toxicity seen in the partner MLA study TL38FR.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 125 μg/mL, in all three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 125 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 31.25 μg/mL. The mitotic index data are presented in Table 1. The test item demonstrated marked toxicity in all three exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 16 μg/mL for both of the 4(20)-hour exposure groups and 2 μg/mL for the 24-hour exposure group. The dose selection for all exposure groups of the main test aimed to achieve a dose level where optimum toxicity was demonstrated.

Chromosome Aberration Test – Main Experiment
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 present at all test item dose levels. In the 4(20)-hour exposure groups in the absence and presence of metabolic activation (S9), the maximum dose level of the test item with metaphases suitable for scoring was 16 μg/mL. In the continuous exposure group the maximum dose level of the test item with sufficient metaphases suitable for scoring was 1 μg/mL, although there were a few metaphases present at 2 μg/mL.
Precipitate observations were made at the end of exposure for all three exposure groups and no precipitate was observed.
The mitotic index data for the Main Experiment are given in Table 2 and Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, optimum toxicity was achieved at 8 μg/mL with 43 % mitotic inhibition. In the 4(20)-hour exposure group in the presence of S9, 31 % and 71 % mitotic inhibition was achieved at 8 and 16 μg/mL, respectively. Although mitotic inhibition of 71% exceeded optimum toxicity it was selected for scoring as the lower dose level of 8 μg/mL demonstrated insufficient toxicity. The 24-hour exposure group achieved near optimum toxicity at 1 μg/mL with 54 % mitotic inhibition. The dose level of 2 μg/mL in the 24-hour exposure group was too toxic for scoring and had insufficient metaphases for evaluation.
The maximum dose level selected for metaphase analysis was 8 μg/mL in the 4(20)-hour exposure group in the absence of S9, 16 μg/mL in the 4(20)-hour exposure group in the presence of S9 and 1 μg/mL in the 24-hour continuous exposure group.
The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analyzed.
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 any of the three exposure groups.

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

Exposure Group	Final concentration ofPigment Red 81:4 (µg/mL)
4(20)-hour without S9	0*, 0.25, 0.5, 1*, 2*, 4*, 8*, 16, MMC0.2*
4(20)-hour with S9 (2%)	0*, 0.25, 0.5, 1, 2*, 4*, 8*, 16*, CP2*
24-hour without S9	0*, 0.031, 0.063, 0.125*, 0.25*, 0.5*, 1*, 2, MMC0.2*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Conclusions:

Pigment Red 81:4 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 metabolizing system. 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., 1991).

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were as follows:

Exposure Group	Final concentration of test itemPigmentRed 81:4(µg/mL)
4(20)-hour without S9	0, 0.25, 0.5, 1, 2, 4, 8, 16
4(20)-hour with S9 (2%)	0, 0.25, 0.5, 1, 2, 4, 8, 16
24-hour without S9	0, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2

Results

All vehicle (dimethyl sulphoxide) 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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was markedly toxic but did not induce any statistically significant increases in the frequency of cells with aberrations. The dose range included a dose level that induced approximately 55±5% mitotic inhibition or greater.

Conclusion

The test item, Pigment Red 81:4 was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro gene mutation study in bacterial (Ames study):

Introduction:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre‑incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000  µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate.

Seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Results:

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In Experiment 1 (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the tester strains in both the absence and presence of S9-mix, initially from 1500 µg/plate (TA1537) and at 5000 µg/plate (remaining strains). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second experiment. In Experiment 2 (pre‑incubation method) the test item induced a stronger toxic response with either a visible reduction in the growth of the bacterial background lawn and/or a substantial reduction in the frequency of revertant colonies noted to all of the tester strains, initially from 500 µg/plate (E.coli strain WP2uvrA, TA1537 with and without S9 and TA1535 in the absence of S9‑mix only) and from 1500 µg/plate to the remaining strains. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. In Experiment 1 (plate incorporation method), an intense pink test item induced coloration was noted from 1500 µg/plate with an associated precipitate (powdery in appearance) observed at 5000 mg/plate. In Experiment 2, after the change in test methodology, an intense pink test item induced coloration was only noted at 1500 µg/plate and the associated precipitate was much thicker at 5000 mg/plate. None of these observations prevented the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Small, statistically significant increases in TA1537 revertant colony frequency were noted in the absence of S9‑mix at 150 and 500 µg/plate. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 and 500 µg/plate were within the in‑house historical untreated/vehicle control range for the tester strain. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). 

Conclusion:

The test item was considered to be non-mutagenic under the conditions of this test.

In vitro gene mutation study in bacterial (Chromosome aberration):

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., 1991).

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were as follows:

Exposure Group	Final concentration of test itemPigmentRed 81:4 (µg/mL)
4(20)-hour without S9	0, 0.25, 0.5, 1, 2, 4, 8, 16
4(20)-hour with S9 (2%)	0, 0.25, 0.5, 1, 2, 4, 8, 16
24-hour without S9	0, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2

Results

All vehicle (dimethyl sulphoxide) 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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was markedly toxic but did not induce any statistically significant increases in the frequency of cells with aberrations. The dose range included a dose level that induced approximately 55±5% mitotic inhibition or greater.

Conclusion

The test item, Pigment Red 81:4 was considered to be non-clastogenic to human lymphocytes in vitro.

In vitro gene mutation study in bacterial (L5178Y TK +/- Mouse Lymphoma Assay):

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group	Concentration of Pigment Red 81:4 (µg/mL) plated for viability and mutant frequency
4-hour without S9	1, 2, 4, 8, 16, 32
4-hour with S9 (2%)	2, 4, 8, 16, 32, 64
24-hour without S9	0.03, 0.06, 0.13, 0.25, 0.5, 1

Results

The maximum dose levels in the Mutagenicity Test were limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Justification for selection of genetic toxicity endpoint

The Ames study, chromosome aberration and mouse lymphoma assay were conducted on the test substance according to internationally recognised OECD guidelines. These studies are considered to be of reliability 1.

Justification for classification or non-classification

An in vitro gene mutation study in bacteria (according to OECD 471) showed the substance to be non-mutagenic under the conditions of the test.

A chromosome aberration test (according to OECD 473): the test item to be considered non-clastogenic to human lymphocytes in vitro.

OECD Guideline 473 ( L5178Y TK +/- Mouse Lymphoma Assay): The test item is considered to be non-mutagenic in this assay.

Based on available information the substane is therefore not classified as a germ cell mutagen.