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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2011-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,4,6(1H,3H,5H)-Pyrimidinetrione, 5,5'-(1H-isoindole-
1,3(2H)- diylidene)bis-
- Substance type: Solid, orange
- Physical state: solid
- Analytical purity: > 99g/100g
- Purity test date: 2011
- Lot/batch No.: 081012P040
- Expiration date of the lot/batch: available in the sponsor's files
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other: Date of production: 29 Mar 2008

Method

Target gene:
hprt
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 from rats treated with phenobarbital i.p. and β-naphthoflavone orally
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL

with S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL

2nd Experiment
without S9 mix (24-hour exposure period)
0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00; 400.00 μg/mL

with S9 mix (4-hour exposure period)
0; 3.75; 7.50; 15.00; 30.00; 50.00 μg/mL
Vehicle / solvent:
culture medium (Ham's F12)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 300 μg/mL ethyl methanesulfonate (without S9) or 20 μg/mL methylcholanthrene (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24h
- Exposure duration: 4 hours both with and without metabolic activation and 24 hours without metabolic activation
- Expression time (cells in growth medium): 6 - 8 days
- Selection time (if incubation with a selection agent): 1 week
- Fixation time (start of exposure up to fixation or harvest of cells): 14 - 16 days

SELECTION AGENT (mutation assays): 6-thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
pH
Changes in the pH were recorded by a change in the indicator color in the culture medium
(phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two
top doses and for the negative controls with and without S9 mix.

Osmolarity
Osmolarity was measured, at least for the top dose and for the negative controls with and
without S9 mix.

Solubility
Test substance precipitation was checked immediately after treatment of the test cultures and
at the end of treatment.

Cell morphology
The test cultures of all test groups were examined microscopically at the end of exposure
period with regard to cell morphology, which allows conclusions to be drawn about the
attachment of the cells.
Evaluation criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than
50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our
historical negative control data range of 0 – 15.95 mutants per 106 clonable cells (see
Appendix 5).
• The positive controls both with and without S9 mix must induce distinctly increased
mutant frequencies (historical positive control data; see Appendix 6).
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of
solubility under culture conditions should be tested. Freely soluble and apparently
non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent
negative control values and our historical negative control data range (see Appendix 5).
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse
relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e.
15 mutants per 106 clonable cells) or isolated statistically significant increases without a
dose-response relationship may indicate a biological effect but are not regarded as sufficient
evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant
increased above the concurrent negative control and is within our historical negative
control data range.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
The test substance was poorly soluble in the most suitable vehicle culture medium.
Due to lacking distinct cytotoxicity in the pretests in the absence and presence of metabolic
activation after 4 hours exposure all experimental parts with short-term exposure were
performed using concentrations at the border of saturation of culture medium.
In the pretest with 24 hours exposure in the absence of metabolic activation cytotoxicity of
about 20% relative survival was observed at intermediate concentrations showing strong test
substance precipitation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

A table with the results is attached as a jpg file.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative