Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, sulfonated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: Test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity - Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471. Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of 15 - 2500 µg which were applied to plates in volume of 0.1 mL. Two series of experiments were performed with each strain, without metabolic activation and with a supernatant of rat liver and a mixture of cofactors. For all used strains was substance with and without metabolic activation non mutagenic.

 

Mouse Lymphoma: 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 Guidelines 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, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

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 (R0 medium), 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: 0.16, 0.31, 0.63, 1.25, 2.5, 5 µg/mL. The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. The vehicle control cultures had mutant frequency values that were 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. 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.

 

Human lymphocytes: The study was to assess the potential chromosomal mutagenicity of the test item on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test. All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) 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 non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

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:

23.08.2013 - 11.10.2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see Deviations in the Section: Additional information on results

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan dependence

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan dependent strain

Metabolic activation:

with and without

Metabolic activation system:

a supernatant of rat liver and a mixture of cofactors

Test concentrations with justification for top dose:

First round of experiments: 50, 150, 500, 1500 and 5000 µg per plate
Second round of experiments: 15, 50, 150, 500, 1500 µg per plate
In first round of experiments in 5000 µg per plate decrease of number of revertants occured.

Vehicle / solvent:

- Vehicle: water
- Justification for choice of vehicle: optimal solvent for test substance

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine hydrochloride monohydrate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

THE BACTERIAL TESTER STRAINS:
- histidine dependent Salmonella typhimurium TA 98 (CCM 3811), TA 100 (CCM 3812), TA 1535 (CCM 3814), TA 1537 (CCM 3815) and tryptophan dependent strain Escherichia coli WP2 uvrA (CCM 4751) were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno,

Strains TA 1537 and TA 98 detect frame shift mutations,
strains TA 100 and TA 1535 serve to detect base-pair substitution mutations,
strain E.coli WP2 uvrA detects cross-linking mutagens.

METHOD OF APPLICATION: in agar (plate incorporation)

Plate incorporation test
Test procedure:
100 µL of the test substance in required concentration, 0.1 mL 16-18 h culture of tester strain, 0.5 mL relevant buffer and 30 or 100 µL of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3ºC. After shaking the mixture was poured into a minimal glucose agar plate. After incubation of 48 - 72 h at 37±1ºC, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted automatically.
For an adequate estimate of variation, triplicate plating was used at each dose level.

Selection of doses/toxicity:
The test substance was suspended in water in concentration 2 500 microgram/0.1 mL. Starting suspension was diluted to concentration series (10 - 2500 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. Besides, maximum dose recomended by guidelines - 5000 µg per plate - was dosed in volume 0.2 ml. Applications suspensions were shaken before withdrawal of aliquots for diluting as well as before
pouring of top agar with the test substances onto Petri dishes.
Precipitation was observed in dishes from the dose of 500 µg per plate, but no toxicity or difficulties with evaluation were observed during the toxicity experiment.

Dose of 5000 µg per plate was then used as maximum in the first mutagenicity experiments. The starting concentration was diluted according to guidelines (five different analysable concentrations with approximately half log (i.e. √10) intervals between test points). In the second experiments was used maximum dose 2500 µg per plate and additional dose of15 µg per plate in experiments.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as "biologically relevant":
-if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversions more than 10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤ 10.
According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of resukts, a statistical evaluation of te results is noit regarded as necessary.

Statistics:

According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of resukts, a statistical evaluation of te results is not regarded as necessary.

Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and 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:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Deviations:
Two more experiments were performed in addition to the planned ones. In the second experiments in TA 98 (with and without metabolic activation), bacterial suspension used for testing was contaminated, so Petri dishes could not be evaluated. These experiments were repeated. This deviation had no impact onthe outcome of the study.

see attached background document

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation for all used strains
negative without metabolic activation for all used strains

Executive summary:

Test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity - Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dosed at 15 - 2500 µg which applied to plates at a volume of 0.1 mL.

Two series of experiments were performed with each strain, without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

For all used strains was substance with and without metabolic activation non mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2017 to 23 May 2017

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 using the Thymidine Kinase Gene

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Suitability of cells: Suitable for study
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not reported
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

2% S9

Test concentrations with justification for top dose:

Eight dose levels of the test item (0.16 to 20 µg/mL for all three of the exposure groups), vehicle and positive controls.

The test item was considered to be a complex mixture (UVCB) therefore the maximum proposed dose level in the solubility test was set at 5000 µg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. However, the test item was not suitable for dosing at 500 mg/mL. Therefore, the test item was formulated at 250 mg/mL and dosed at 10% to give a maximum achievable dose level of 2500 µg/mL in the subsequent preliminary toxicity test. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and osmolality readings from the Chromosome Aberration Test are in the following table (located in the "Any other information on materials and methods incl. tables" section).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0 medium) exposure groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) - Microtitre plates.
- Cell density at seeding (if applicable): 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. The dose range used in the preliminary toxicity test was 9.77 to 2500 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 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. For the mutagenicity test, 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.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 or 24 hours hours
- Expression time (cells in growth medium): 2 days

STAIN: Thiazolyl blue tetrazolium bromide (MTT) solution

NUMBER OF REPLICATIONS: Performed in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Thiazolyl blue tetrazolium bromide (MTT) solution

DETERMINATION OF CYTOTOXICITY
- Method: On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

Rationale for test conditions:

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. 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).

Evaluation criteria:

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.

Statistics:

See attached background document for equations.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Positive controls validity:

valid

Additional information on results:

There was no evidence of any marked toxicity following exposure to the test item in any of the three exposure groups, as indicated by the %RSG and RTG values. There was also no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances.

Unexpectedly, precipitate of the test item was observed at all of the dose levels in the 4-hour exposure groups, and at and above 0.31 µg/mL in the 24-hour exposure group, at the end of the exposure periods. However, it was considered that a maximum dose level lower than 0.16 µg/mL would be beyond the accuracy of the pipettors and this study type. It should also be noted that there was no evidence of any response at the one non-precipitating dose level in the 24-hour exposure group. Therefore, the test item was considered to have been adequately tested. The upper two dose levels of 10 and 20 µg/mL were not plated as they were considered to be surplus to requirements with precipitate present at the lower dose levels. 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 (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels in any of the three exposure groups.

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 9.77 to 2500 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (µg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
9.77	85	100	96
19.53	84	105	103
39.06	84	100	110
78.13	86	103	92
156.25	44	73	33
312.5	49	79	4
625	50	58	1
1250	49	58	0
2500	46	6	0

Summary of results (Main Experiment):

Concentration (µg/mL)	4-Hour (-S9)			Concentration (µg/mL)	4-Hours (+S9)			Concentration (µg/mL)	24-Hours (-S9)
	%RSG	RTG	MF§		%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	1.88.96	0	100	1.00	129.71	0	100	1.00	165.68
0.16	111	1.21	157.79	0.16	101	0.95	141.21	0.16	96	0.99	145.39
0.31	102	1.26	129.25	0.31	97	0.92	118.29	0.31	104	1.05	142.67
0.63	102	1.09	171.05	0.63	96	1.02	112.53	0.63	89	1.09	148.41
1.25	104	1.26	139.64	1.25	100	0.99	116.7	1.25	92	1.03	149.46
2.5	95	1.09	178.72	2.5	96	0.96	108.21	2.5	87	0.93	142.45
5	91	1.07	154.27	5	97	1.13	91.52	5	128	1.21	160.86
10 Ø				10 Ø				10 Ø
20 Ø				20 Ø				20 Ø
MR threshold for a positive response = 314.96				MF threshold for a positive response = 255.71				MR threshold for a positive response = 291.68
Positive Control				Positive Control				Positive Control
EMS 400	78	0.68	1190	CP 1.5	82	0.57	871.86	EMS 150	43	0.39	1601.92
Notes: MF§ = 5-TFT resistant mutants/106 viable cells 2 days after exposure Ø = Not plated due to the presence of precipitate

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:

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 Guidelines 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, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

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 (R0 medium), 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: 0.16, 0.31, 0.63, 1.25, 2.5, 5 µg/mL.

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. The vehicle control cultures had mutant frequency values that were 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.

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:

11 April 2017 to 21 July 2017

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Species / strain / cell type:

lymphocytes: Whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability.

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human volunteers
- Suitability of cells: screened for suitability
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: Preliminary Toxicity Test: male, aged 27 years; Main Experiment: female, aged 26 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 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
- Properly maintained: not reported
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically 'cleansed' against high spontaneous background:not reported

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The dose range of test item for all three exposure groups used was 0.063, 0.13, 0.25, 0.5, 1, 2 and 4 µg/mL.

Vehicle / solvent:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): Not reported

DURATION
- Preincubation period: Not reported
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SELECTION AGENT (mutation assays): Not applicable

SPINDLE INHIBITOR (cytogenetic assays): Not reported

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: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 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 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.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 1.5 x AGT is 24 hours.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:

- OTHER: Scoring of Chromosome Damage:
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. 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 and the ISCN (1985) (Appendix 1). 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 (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Rationale for test conditions:

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, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 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 test.

Evaluation criteria:

The following criteria were used to determine a valid assay:

• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment 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

Statistics:

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.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: Whole blood from human volunteers

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:

valid

Positive controls validity:

valid

Conclusions:

The test substance 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:

The study was to assess the potential chromosomal mutagenicity of the test item on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test. All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) 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 non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available