Disodium tin hexahydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 2008 to 02 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description: white powder

Method

Target gene:

TK gene

Species / strain

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 -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) and without serum (R0) are used during the course of the study.


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.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β naphthoflavone activated rat liver S9

Test concentrations with justification for top dose:

Concentrations used in the preliminary toxicity test (with and without metabolic activation)
0, 11.17, 22.34, 44.69, 89.38, 178.75, 357.5, 715, 1430, 2860 µg/ml

Concentrations used in the main test (with and without metabolic activation)
0, 178.75, 357.5, 715, 1430, 2145, 2860 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The test material was dissolved in DMSO prior to dilution to the test concentrations.
- Justification for choice of solvent/vehicle: Not reported.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "Any other information on materials and methods inc. tables" section

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period:
- Exposure duration: 4 hours (in both the presence and absence of S9) and 24hours (in the absence of S9 only)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 52 to 72 hours

SPINDLE INHIBITOR (cytogenetic assays): 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): MTT

NUMBER OF REPLICATIONS: 2 per experiment

NUMBER OF CELLS EVALUATED: ca. 192000 (2000 cells/well, 96 wells)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002. It was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 at 126 x 10.6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive.

However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS

Calculation of % Relative Suspension Growth (%RSG)
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the % Relative Suspension Growth.

Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Plating Efficiency (PE)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the plating efficiency (PE), from which is derived the viability (%V), was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells/total wells plated
PE% = -ln P(0) x 100/number of cells per well

Calculation of Relative Total Growth (RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = PE/Mean Solvent Control PE x 100%

Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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 applicable

Positive controls validity:

valid

Additional information on results:

The maximum dose level used was the 10 mM limit dose. A precipitate of test material was observed at and above 178.75 µg/ml. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment using a dose range that included the 10 mM limit dose.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1
Treatment (µg/ml)	4-Hours-S9			Treatment (µg/ml)	4-Hours +S9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1	148.68	0	100	1	195.82
178.75	111	1.06	159.2	178.75	123	1.24	160.01
357.5	106	1.16	157.24	357.5	108	1.16	132.92
715	94	1	209.65	715	109	1.18	173.12
1430	80	0.95	132.72	1430	103	1.19	154.93
2145	72	0.81	111.64	2145	97	0.98	168.36
2860	71	0.75	108.05	2860	102	1.26	164.29
Linear trend			NS	Linear trend			NS
EMS				CP
400	83	0.6	646.27	0	61	0.27	992.73
Experiment 2
Treatment (µg/ml)	24-Hours-S9			Treatment (µg/ml)	4-Hours +S9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1	86.16	0	100	1	102.03
178.75	144	1.18	76.1	178.75	122	1.17	69.1
357.5	157	1.34	65.37	357.5	116	1.05	93.45
715	150	1.19	100.71	715	105	1.21	73.23
1430	154	0.99	116.97	1430	97	1	79.94
2145	154	1.33	102.07	2145	102	0.94	82.48
2860	159	1.24	107.49	2860	91	0.99	69.77
Linear trend			*	Linear trend			NS
EMS				CP
150	83	0.48	918.42	0	57	0.33	599.73

 

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

CP = Cyclophosphamide

EMS = Ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/10^6 viable cells 2 days after treatment

* = p<0.05

NS = Not significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative Negative, both in the presence and absence of metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

In a mammalian cell gene mutation assay performed in accordance with OECD Guidelines for the testing of chemicals No. 476 and EU method B17, TK +/- locus, L5178Y cells cultured in vitro were exposed to the test material at concentrations of up to 2860 µg/mL (10 mM limit dose) in both the presence and absence of mammalian metabolic activation

The positive controls induced the appropriate response. The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.