[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human, cultured in vitro in whole blood culture

Details on mammalian cell type (if applicable):

CELLS USED
- Source of lymphocytes: Human blood collected aseptically from two healthy, non-smoking male donors and pooled.
- Normal cell cycle time (negative control): cells do not divide unless stimulated with phytohaemagglutinin. In this laboratory the cell cycle time is approximately 13 - 14 hours.

For lymphocytes:
- Sex, age and number of blood donors: two male, non-smoking donors
- Whether whole blood or separated lymphocytes were used: whole blood was used
- Whether blood from different donors were pooled or not: pooled blood was used
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 supplemented with 10% foetal calf serum, 1 I.U./ml sodium heparin, 20 I.U./ml penicillin / 20 µg/ml streptomycin and 2.0 mM glutamine. Aliquots (0.4 ml blood : 4.5 ml medium : 0.1 ml phytohaemagglutinin) of cell suspension were placed in sterile universal containers and incubated at 37°C for approximately 48 h. The cultures were gently shaken daily to resuspend the cells.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Sprague-Dawley rats (Aroclor 1254 induced rat liver fraction)

Test concentrations with justification for top dose:

EXPERIMENT 1:
Concentrations prepared without and with metabolic activation (S9): 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.

Due to a steep toxic response observed in both the absence and presence of S9 mix at concentrations above 78.13 μg/mL, a repeat test was performed using the following concentrations of T-71: 0, 6.25, 12.5, 25, 50, 75, 100 and 125 μg/mL.

EXPERIMENT 2:
Concentrations of T-71 were as follows:
Without S9 mix: 0, 3.13, 6.25, 12.5, 25, 50 and 75 μg/mL.
With S9 mix: 0, 6.25, 12.5, 25, 50 and 75 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle:
Information provided by the sponsor indicated that dimethyl sulphoxide, purified water and acetone were not suitable solvents. T-71 was also found to be insoluble in ethanol, however a dosable suspension was formed in culture medium at 10 mg/mL. On dosing at 50% v/v into aqueous tissue culture medium, giving a final concentration of 5000 µg/mL, heavy precipitate was observed. In this case, the highest concentration used for subsequent testing was 5000 µg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide tested at 10 μg/mL, vehicle sterile purified water: with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C tested at 0.2 μg/mL (3 hour treatment) and 0.1 μg/mL (continuous treatment), vehicle sterile purified water: without metabolic activation

Details on test system and experimental conditions:

CELL DIVISION STIMULANT:
Phytohaemagglutinin

METHOD OF APPLICATION:
in cell culture medium

DURATION
- Exposure duration:
3 hours in Experiment 1 (without and with metabolic activation) and in Experiment 2 (with metabolic activation).
20 hours in Experiment 2 (without metabolic activation)
[After the 3 h treatment the cells were cultivated with fresh media for 17 h].
- Final concentration of S9 mix:
Experiment 1: cultures contained 1 ml S9 mix and 2.5 mL cell culture medium
Experiment 2: cultures contained 1 ml S9 mix and 2.5 mL cell culture medium
- Fixation time (start of exposure up to fixation or harvest of cells):
20 hours in each of both experiments

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® was added to the cultures (0.1 µg/mL culture medium) 18 hours after treatment start.
2 h later, the cells were treated with hypotonic solution (0.075 M KCl) for 10 min at 37 °C. After incubation in the hypotonic solution, the cells were fixed with 3 + 1 methanol + glacial acetic acid.

STAIN (for cytogenetic assays):
After fixation the cells were stained with 10% Giemsa.

NUMBER OF REPLICATIONS:
Duplicate cultures were treated at each concentration.

NUMBER OF CELLS EVALUATED:
100 metaphases per culture, amounting to a total of 200 metaphases per dose concentration, were scored for structural chromosomal aberrations.
This number was reduced In cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) determined by counting the number of mitotic cells in 1000 cells;

Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (including and excluding gaps),
- Number of gaps,
- Types of aberrations
Chromatid break, Chromosome break, Chromatid gap, Chromatid exchange, Chromosome exchange, Chromosome gap,
Others: Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes

Determination of polyploidy:
- Polyploid and endoreduplicated cells were noted when seen.

Evaluation criteria:

An assay is considered to be acceptable if the vehicle and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
-Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
-The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
-Evidence of a dose-relationship is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.

Statistics:

One-tailed Fisher exact test (Fisher 1973) for comparison of the number of aberrant metaphase cells in each test substance group with the solvent control value.

FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York.

Key result

Species / strain:

lymphocytes: human

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

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments following 3 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With metabolic acitviation and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See table on results 'Table CA NA-70.pdf' attached as background material.

Conclusions:

It is concluded that the test substance T-71 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC Comission Directive 2000/32/EC Annex 4E.B17, No L136/65

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase locus in heterozygous mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Medium R0: RPMI 1640, buffered with sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamycin.

Medium R10p: R0 medium, supplemented with 0.1% v/v Synperonic F68, 0.01 % sodium pyruvate and HiDHS at 10% v/v.

Medium R30p: R0 medium, supplemented with 0.02% v/v Synperonic F68, 0.01 % sodium pyruvate and HiDHS at 30% v/v was mixed with R10p for Day0 relative survival plating.

Selective medium consisted of R10p containing trifluorothymidine (TFT)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254 induced rat liver fraction)

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TESTING (suspension growth relative to that of vehicle controls)
Test concentrations at 3 h exposure with (+S9) and without (–S9) metabolic activation and at 24 h exposure without metabolic activation (–S9):
15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 20, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5 and 50 µg/mL
Mutant phenotype determination at: 20, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5 and 50 µg/mL

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 20, 30, 35, 40, 45, 50, 55 and 60 µg/mL
Mutant phenotype determination at: 20, 30, 35, 40, 45, 50 µg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 10, 20, 30, 32.5, 35, 37.5, 40, 45, and 50 µg/mL
Mutant phenotype determination at: 10, 20, 30, 32.5, 35, 37.5, 40 and 45 µg/mL

Experiment 2, 3 h exposure (+S9):
Exposure concentrations: 20, 30, 35, 37.5, 40, 42.5, 45, 47.5 and 50 µg/mL
Mutant phenotype determination at: 20, 30, 35, 37.5, 40, 42.5, 45, 47.5 and 50 µg/mL

Experiment 3, 24 h exposure (-S9):
Exposure concentrations: 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5 and 50 µg/mL
Mutant phenotype determination at: 30, 32.5, 35, 37.5, 40, 42.5 and 45 µg/mL


CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (i.e. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: the test material was dissolved and diluted in DMSO shortly before dosing. the final concentration of DMSO added to the cultures was 1% v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

Positive control substance for tests with metabolic activation (-S9). 2.5 µg/mL, vehicle DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control substance for tests without metabolic activation (-S9). 3h exposure: 10 µg/mL; 24 h exposure: 5 µg/mL, vehicle DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment 1: 3 h exposure with (+S9) and without (–S9) metabolic activation
Experiment 2: 3 h exposure with (+S9) and 24 h exposure without metabolic activation (–S9)
Experiment 3: 24 h exposure without metabolic activation (–S9)

- Selection time: At 48 h after the end of exposure addition of the selection agent trifluorothymidine (TFT)
then allowing 10-14 days for cells to grow with TFT.

SELECTION AGENT: Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 cultures at each concentration,
[from each culture two vials for assessment of growth in suspension, two 96-well plates for assessment of cloning efficiency
and two 96-well plates for assessment of mutant potential].

NUMBER OF CELLS EVALUATED: 2000 cell/well x 192 wells = 384000 cells per culture

DETERMINATION OF CYTOTOXICITY: Relative total growth

Evaluation criteria:

The statistical significance of the data was analysed by following the methods described by Robinson et al. (1989). The criteria for a positive response were:

The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance.

Evidence of a dose relationship over at least two dose levels, in any increases in mutant frequency.

Demonstration of reproducibility in any increases in mutant frequency.

Any observed increases in mutant frequency should lie outside the upper 95 % confidence limits for the historical solvent control range with a corresponding mean Day0 RS values of not less than 10%.

Statistics:

The data were analysed using the methods described by Robinson et al. (1989).

Robinson, W.D., Green, M.H.L., Cole, J., Healy, M.J.R., Garner, R.C., and Gatehouse, D. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: Kirkland, D. J. (Ed). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part 111. Statistical Evaluation of Mutagenicity Test Data, p.102-140. Cambridge University Press, Cambridge.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment 3, 24h (-S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Experiment 2, 3h exposure (+S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment 2, 24h exposure (-S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

Preliminary Toxicity Testing: 3 h exposure (–/+S9) and 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Detailed in Table 1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiment 1, 3 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Experiment 1, 3h exposure (+S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The concentrations chosen for mutagenicity testing In both main mutation experiments (Experiments 1 and 2) were considerably lower than 2000 µg/mL because of cytotoxicity (detailed in Table 1).

See tables on MLA results 'Tables MLA L5178Y_NA-70.pdf' attached as background material.

Conclusions:

NA-70 demonstrated mutagenic potential after exposure for 24 hours in the absence of S9 mix in this in vitro cell mutation assay, under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC Comission Directive 2000/32/EC Annex 4D-B.13.14, No L136/57

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus in selected S. typhimurium strains. Tryptophan locus in selected E. coli strain.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Aroclor 1254 induced rat liver fraction)

Test concentrations with justification for top dose:

Range finding: seven concentrations up to 5000 µg/plate
Main test: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water containing 0.15% bacteriological agar (Oxoid)
- Justification for choice of solvent/vehicle: the test material was found to be insufficiently soluble in water and insoluble in dimethyl sulfoxide.

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Benzo(a)pyrene for strains TA 1537, TA 98 and TA100 with metabolic activation, dissolved in DMSO, 5 µg/plate.

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (CAS No: 613-13-8)

Remarks:

2-Aminoanthracene for strain TA 1535 with metabolic activation, dissolved in DMSO, 2 µg/plate; for strain WP2uvrA/pKM101 with metabolic activation, dissolved in DMSO, 10 µg/plate.

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

furylfuramide for strains WP2uvrA/pKM101 without metabolic activation, dissolved in DMSO, 0.05 µg/plate.

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2-nitrofluorene for strain TA 98 without metabolic activation, dissolved in DMSO, 1 µg/plate.

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

9-aminoacridine for strain TA 1537 without metabolic activation, dissolved in DMSO, 50 µg/plate.

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent controls were perfomed

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

sodium azide for strains: TA 1535, TA 100 without metabolic activation, 0.5 µg/plate, dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Preincubation period: 30 min., in glass vessels, with test material in solution
- Incubation time: 72 hours (petri dishes containing minimal agar without tryptophan)

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY in the range finding test
- Method: substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

The mutagenic activity of a test substance was assessed by applying the following criteria:
- increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-response relationship (...)
- If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

Statistics:

The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett's test.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Interpretation of results : negative

It is concluded that, under the test conditions employed, the test material T-71 showed no evidence of mutagenic activity in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

21. July 1997

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Version / remarks:

19. May 2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the UDS test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Wistar HanIbm: WIST (SPF)
- Source: RCC Ltd, Animal Breeding Services, CH-4414 Füllinsdorf
- Weight at study initiation: 183.7 ± 15.5 (SD) g (males)
- Number of animals: 32 (males)
- Assigned to test groups randomly: yes
- Housing: single housing
- Diet (e.g. ad libitum): pelleted standard diet (Altromin) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12; artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% w/v sodium carboxymethylcellulose (CMC)
- Concentration of test material in vehicle: 0, 1000, 2000 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw (10 mL/kg bw for positive control substances).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.5% CMC (Fluka, Cat. no: 21902). The vehicle was chosen to its non-toxicity for the animals. The animals received a single standard volume of 20 ml/kg body weight orally (the positive control groups were treated with 10 mL/kg body weight).

Duration of treatment / exposure:

The test item was administered once for 2 h or or 16 h.

Frequency of treatment:

single treatment

Post exposure period:

2 h and 16 h after dosing.

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 males per dose group (main experiment).
2 female and 2 male per dose (pre-test).

Control animals:

yes, concurrent vehicle

Positive control(s):

- 4 male rats administered N,N'-dimethylhydrazinedihydrochloride (sym.) (DMH) (40 mg/kg bw) in 0.9 % NaCl solution (for 2 hours preparation interval).

- 4 male rats administered 2-acetylaminofluorene (2-AAF) (100 mg/kg bw) in dimethylsulfoxide/polyethylene glycol 400 (1 + 9) (for 16 hours preparation interval).

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Preliminary study at max. dose of 2000 mg/kg body weight in 2 male and female rats

TREATMENT
The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test item, the positive or vehicle control substance. Water was available ad libitum. Animals were weighed and the volume to be administered was adjusted to the body weight of each animal. The animals received the test item once. Four animals (males) were treated per group.

ISOLATION OF PRIMARY HEPATOCYTES
After anesthesizing the rats with Na-thiopental (Trapanal, Byk Gulden) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL) supplemented with 0.05 % w/v collagenase (Boehringer Mannheim) adjusted to pH 7.4 and maintained at 37 °C.

The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh (94 µm) to yield a single cell suspension. Cell viability was determined using trypan blue dye exclusion. In addition, the number of cells was determined.

CULTURE CONDITIONS
The washed cells were centrifuged and transferred into Williams medium E (WME, Gibco/BRL) supplemented with Hepes (2.38 mg/mL), Penicillin (100 units/mL), Streptomycin (0.10 mg/mL), L-Glutamine (0.29 mg/mL), Insulin (0.5 µg/mL) and Fetal calf serum (FCS, 100 µl/mL). This complete medium was adjusted to pH 7.6.

At least three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0 x 10^5 viable cells/mL) were added to 35 mm six-well dishes (Greiner) containing one 25 mm round plastic coverslip (Thermanox) per well coated with gelatine.

After 1.5 h in a 95 % air / 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 20 Ci/mmol; New England Nuclear) in 2.0 mL culture medium (WME, 1 % v/v FCS) was added to the cultures. After a labelling for 4 h, cells were washed twice with WME, 1 % v/v FCS and 0.25 mM unlabelled thymidine and incubated overnight. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3 + 1 v/v) for 20 min each, rinsed with 96 % (v/v) ethanol, and air-dried.

AUTORADIOGRAPHIC PROCESSING
Coverslips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Tecnomara) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4° C. The photographic emulsion was then developped with Ilford Phenisol (Ilford Imaging) at room temperature, fixed with Rapid Fixer (Ilford Imaging) and stained with hematoxylin/eosin.

QUANTIFICATION OF UDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. Cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labeled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily radiolabelled cells undergoing replicative DNA synthesis were excluded from counting.

Three animals per group were evaluated as described above. The remaining animal per test group would have been evaluated if an animal died spontaneously or in case of technical problems concerning the isolation of hepatocytes. This was not the case in the present study.

METHOD OF ANALYSIS: light microscopy

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.

A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.

A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the
percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.

Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A test item producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The treated animals expressed toxic reactions such as a reduction of spontaneous activity (2/4), ruffled fur (4/4) and eyelid closure (0/1) 2 h after treatment with 1000 / 2000 mg/kg b.w. of T-71.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

see attached "Tables 10.4 Mean Nucleus, Cytoplasmic Area, and Net Grains" for details of results.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

EC Comission Directive 2000/32/EC Annex 4C - B.12, No L136/50

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd
- Weight at study initiation: 28-32 g (males), 22 - 26 g (females)
- Assigned to test groups randomly: yes
- Housing: group housing, sexes separated
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% w/v methylcellulose
- Concentration of test material in vehicle: 0, 25, 50, 100 mg/ml
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: mixing of T-71 with 1% w/v methylcellulose (Aldrich, batch number 02206TF)

Duration of treatment / exposure:

The test item was administered once.

Frequency of treatment:

single treatment

Post exposure period:

24 h (all groups) and 48 h (vehicle and 2000 mg/kg bw/day groups) after dosing.

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

7 males per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

5 male mice administered Mitomycin C (12 mg/kg bw/day)

Tissues and cell types examined:

bone marrow from femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: preliminary study at max. dose of 2000 mg/kg/day in male and female mice

DETAILS OF SLIDE PREPARATION:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in 10% Giemsa solution (prepared by 1:9 dilution of Giemsa with purified water) solution for 10 minutes
4 Rinsed in purified water
5 Differentiated in buffered purified water
6 Rinsed in purified water and allowed to air-dry
7 Slides were mounted with coverslips using DPX

METHOD OF ANALYSIS: light microscopy

Evaluation criteria:

Acceptance Criteria

The following criteria were applied for assessment of assay acceptability:

The treated and control groups had included at least five animals per sex.
The proportion of immature erythrocytes among total erythrocytes in treated groups was not less than 20% of the control value.

A positive result is normally indicate by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatmenent group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical range (Morrison and Ashby 1995).

A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range.

An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01).

Statistics:

The results of each treatment group were compared with the results for the concurrent control group using non-parametric statistics.

For incidences of micronucleated immature erythrocytes, exact one-sided P-values are calculated by permutation (StatXact, CYTEL Software Corporation, NC, USA). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (StatXact, CYTEL Software Corporation, NC, USA); for individual inter-group comparisons (ie the positive control group) this procedure simplifies to a straitforward permutation test (Agresti et al. 1990, Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see attached "Table 2 and 3 Results for individual animals" for details of results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available experimental test data are reliable and suitable for the purpose of classification according to European Regulation (EC) No. 1272/2008. Based on the data, classification is not warranted.