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Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Cited as Directive 2000/32/EC, B.17
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test compound: Ammonium thioglycolate 71%
Source: Bruno Bock Chemische Fabrik GmbH & Co KG
Batch: 8215
Purity: 71.1% in water
Solvent: deionised water.

Method

Target gene:
Locus Examined: thymidine kynase, the selection agent used was 5 µg/ml trifluorothymidine
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: the medium used was RPMI 1640 (complete medium) with 3 or 15 % horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from male Sprague Dawley (phenobarbital/B-naphtoflavone induced rat liver)
Test concentrations with justification for top dose:
Non-activated conditions: Initial trial: 13, 50, 100, 200, 400, 800 and 1600 µg/mL; Confirmatory trial: 13, 25, 50, 100, 200, 400 and 600 µg/mL
S9-activated conditions: 50, 100, 200, 400, 800 and 1600 µg/mL
Duplicate cultures were processed for all trials.
Vehicle / solvent:
- Solvent used: deionised water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methyl methanesulfonate (13 µg/mL) without activation; cyclophosphamide with activation (3 µg/mL)
Details on test system and experimental conditions:
Experimental Performance:
In the mutation experiment 1x10e7 cells/flask suspended in 10 ml RPMI medium with 3 % horse serum (15 % during 24 h treatment) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation.

After 4 h (after 24 h in the second experiment) the test item was removed by centrifugation and washing twice in "saline G". Subsequently the cells were resuspended in 30 ml complete culture medium and incubated for an expression and growth period of totally 72 h. In the second experiment the expression time without metabolic activation was 48 hours (RPMI medium with 15 % horse serum).

The cell density was determined each day and adjusted to 3x10e5 cells/ml, if necessary. Relative suspension and total growth (RSG and RTG) of the treated tell cultures were calculated after 48 h (72 h following continuous treatment) according to the method of Clive and Spector. One sample of the cells was taken at the end of the treatment (4 h and 24 h, respectively), diluted and seeded into microtiter plates (about 2.5 cells/well), to determine the survival of the cells after treatment (cloning efficiency 1).

After the expression period the tells were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x10e3 tells in selective medium with TFT. The viability (cloning efficiency 2) was determined by seeding about 2.5 cells per well into 2 microtiter plates (same medium without TFT). The plates are incubated at 37 °C in 4.5 %C02 and 95.5 % humidified air for 10 - 15 days to determine the cloning efficiency and to evaluate mutagenicity. Then the plates were evaluated manually.


Size Distribution of the Colonies:
The numbers of colonies were counted manually. The colony size distribution was determined in the controls and at all concentrations of the test item


Data Recording:
All plates were evaluated manually.
The mutation frequency was derived from the cloning efficiency under selective conditions compared to the corresponding viability under non-selective conditions
Evaluation criteria:
Acceptability of the Assay:
A mutation assay is considered acceptable if it meets the following criteria:
a) Both plates, from either the survival or the TFT resistance-testing portion of the experiment are analysable.
b) The absolute cloning efficiency 2 of the negative and/or solvent controls is > 0.5 (50 %).
c) The spontaneous mutant frequency in the negative and/or solvent controls are in the range of the historical control data .
d) The positive controls (MMS and CPA) induce significant (at least 2-fold) increases in the mutant frequencies. The values of the cloning efficiencies and the relative total growth are greater than 10 % of the concurrent vehicle control group.

Evolution of results:
A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points
Statistics:
The survival rate and viability was determined based on the Poisson distribution method. The zero term of the Poisson distribution, [P(0)] method, was used.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 4-h experiment (+/-S9): > 1600 µg/ml / 24-h experiment (-S9): >= 800 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Ammonium Thioglycolate did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

Ammonium thioglycolate was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to the Directive 2000/32/EC, B17 and in compliance with the Principles of Good Laboratory Practice.

In a mammalian cell gene mutation assay (TK+/-), mouse lymphoma L5178Y cells cultured in vitro were exposed to ammonium thioglycolate (purity 71.1%) in deionised water. Two parallels culture were used. The first main experiment was performed without microsomal activation at concentrations of 0, 13, 50, 100, 200, 400, 800 and 1600 µg/ml, and with activation at concentrations of 0, 50, 100, 200, 400,800 and 1600 µg/ml

with a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation at concentrations of 0, 13, 25, 50, 100, 200, 400 and 600 µg/ml, with a treatment period of 24 h. Appropriate positive controls were used and showed a statistical increase in mutant colonies, indicating that the tests were sensitive and valid.

After a 48 rest period, cells were incubated mutagenicity evaluation with trifluorothymidine (TFT).

No substantial and reproductible dose dependant increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Under these experimental conditions, ammonium thioglycolate did not induce any increase in mutant colonies and is not considered as mutagenic in this mouse lymphoma mouse.