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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:
08 July - 11 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Polyglycerol-3
- Substance type: organic liquid
- Physical state: Colourless to pale yellow viscous liquid
- Analytical purity:
27.9% Diglycerol
46.0% Triglycerol
17.9% Tetraglycerol
5.6% Pentaglycerol
2.6% Hexaglycerol and higher
- Purity test date: no data
- Lot/batch No.: RBA081008A
- Expiration date of the lot/batch: 31 May 2010
- Stability under test conditions: stabile

Method

Target gene:
forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: exposure medium
Exposure medium
For 3 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Dose range finding test: 100 to 5000 µg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period
First mutagenicity test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/ml exposure medium (in the absence and presence of 8% (v/v) S9-mix)
Second mutagenicity test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/ml exposure medium (in the absence and presence of 12% (v/v) S9-mix)
Vehicle / solvent:
The vehicle for the test substance was exposure medium. The test substance was dissolved in exposure medium and filter (0.22 µm)-sterilized. Polyglycerol-3 concentrations were used within 45 minutes after preparation.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Exposure medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: (MMS); CAS no. 66-27-3; without metabolic activation (-S9-mix)
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: (CP); CAS no. 50-18-0; With metabolic activation (+S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10-4 M hypoxanthine, 2 x 10-7 M aminopterine and 1.6 x 10-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

- Exposure duration: 3 or 24 h
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period.
- Selection time (if incubation with a selection agent): 11-15 days


SELECTION AGENT (mutation assays): 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/ml trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)


NUMBER OF REPLICATIONS: The test substance will be tested both in the absence and in the presence of S9-mix in two independent experiments.


DETERMINATION OF CYTOTOXICITY
cloning efficiency
mutation frequency
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more then MF(controls) + 126 in a dose-dependent manner; or
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
no

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no


RANGE-FINDING/SCREENING STUDIES: In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 5000 μg/ml compared to the solvent control.


COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent control cultures were between the minimum and maximum value of the historical control data range. Although the mutation frequency of one of the solvent control cultures in the second experiment observed in the absence of S9-mix was just above the historical control data range, the observed mutation frequency of this solvent control culture was within the acceptability criteria of this assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutation frequencies in cultures treated with positive control chemicals were increased by 19- and 9.4-fold forin the absence of S9-mix, and by 16- and 10-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, Polyglycerol-3 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, Polyglycerol-3 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic activity of Polyglycerol-3 was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (in the absence and presence of S9-mix with independent repeat). Polyglycerol-3 was tested up to concentrations of 5000 µg/ml in the absence and presence of S9-mix in the first and second experiment respectively. In the mutation assays, no toxicity was observed and Polyglycerol-3 was tested up to the recommended top concentration of 5000 µg/ml. In the absence of S9-mix, Polyglycerol-3 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.
In the presence of S9-mix, Polyglycerol-3 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.
It is concluded that Polyglycerol-3 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.