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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
Study period:
2011-09-12 to 2011-11-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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
Qualifier:
according to guideline
Guideline:
other: IWGT Recommendations
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dihydro-3-(tripropenyl)furan-2,5-dione
EC Number:
295-556-6
EC Name:
Dihydro-3-(tripropenyl)furan-2,5-dione
Cas Number:
92077-08-2
Molecular formula:
R-C4H3O3 , whereas R=C8-C10-alkyl-(branched, unsaturated)
IUPAC Name:
3-[(1E)-2-methyloct-1-en-1-yl]oxolane-2,5-dione
Details on test material:
Name: Tripropenyl succinic anhydride
Product: Genopur ASA
CAS No.: 92077-08-2
Batch No.: ESD0010823
Certificate of Analysis: dated from 10.09.2011 (see appendix)
Chemical Name: dihydro-3-(tripropenyl)furan-2,5-dione
Molecular Weight: 220.26 g/mol
pH: 1-2
Physical State at RT: liquid
Density: 1.01 g/cm3 (20°C, DIN 51757)
Colour: yellow to brown
Expiry Date: 01.12.2012
Storage Conditions: at room temperature

Method

Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment I with and without metabolic activation: 0.1, 0.5, 2.5, 5.0, 7.5, 10.0 mM
Pre-experiment II without metabolic activation (24 h long-term exposure): 0.1, 0.5, 1.0, 2.0, 4.0, 6.0 mM

Experiment I
without metabolic activation: 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.5, 4.0 mM
with metabolic activation: 0.5, 1.0, 2.0, 3.5, 4.0, 4.5, 5.0, 5.5 mM
Experiment II
without metabolic activation: 0.1, 0.2, 0.5, 1.1, 1.4, 1.7, 2.3, 2.6 mM
with metabolic activation: 1.5, 2.5, 3.7, 4.4, 4.8, 5.2, 5.6, 6.0 mM






Vehicle / solvent:
RPMI cell culture medium was used as vehicle (RPMI + 5% HS)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 2.5 µg/ml
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 200 µg/mL and 300 µg/mL
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:
The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Tripropenyl succinic anhydride is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The test item Tripropenyl succinic anhydride was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 4.0 mM (without metabolic activation) and 5.5 mM (with metabolic activation) were selected as the highest concentrations. In experiment II 2.6 mM (without metabolic activation) and 6.0 mM (with metabolic activation) were selected as the highest concentrations. Experiment I without and with metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay. The test item was suspended in cell culture medium (RPMI with 5% HS)

The test item was investigated at the following concentrations:

Experiment I

without metabolic activation:

0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.5, 4.0 mM

and with metabolic activation:

0.5, 1.0, 2.0, 3.5, 4.0, 4.5, 5.0, 5.5 mM

Experiment II

without metabolic activation:

0.1, 0.2, 0.5, 1.1, 1.4, 1.7, 2.3, 2.6 mM

and with metabolic activation:

1.5, 2.5, 3.7, 4.4, 4.8, 5.2, 5.6, 6.0 mM

No precipitation of the test item was noted in the experiments.

Growth inhibition was observed in experiment I and II without and with metabolic activation.

In experiment I without metabolic activation the relative total growth (RTG) was 4.0% for the highest concentration (4.0 mM) evaluated. The highest concentration evaluated with metabolic activation was 5.5 mM with a RTG of 11.0%. In experiment II without metabolic activation the relative total growth (RTG) was 17.0% for the highest concentration (2.6 mM) The highest concentration evaluated with metabolic activation was 6.0 mM with a RTG of 9.6%. Dose groups showing extremely high toxicity (RTG<10%) were not considered for evaluation of mutagenicity.

The Global Evaluation Factor was not exceeded by the induced mutant frequencies at any concentration (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories). In experiment I with metabolic activation an increased mutation frequency was found in the highest concentration (5.5 mM).This effect was found only in one concentrations in the highly cytotoxic dose range (RTG of 11%) and was not confirmed in the second experiment. This increase in mutant frequency was considered as not biologically relevant.

No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisfies the requirements for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.