Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2011-11-07 to 2012-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
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
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:
sodium/triethanolamine 4-((2-hydroxyethyl)amino)-3-pentaproenyl-4-oxobutanoate
EC Number:
800-765-8
Cas Number:
1424149-03-0
Molecular formula:
C21H40NO4.1/2Na.1/2C6H15NO3
IUPAC Name:
sodium/triethanolamine 4-((2-hydroxyethyl)amino)-3-pentaproenyl-4-oxobutanoate
Details on test material:
Name: Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts
Chemical Name: Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts
Batch No.: ESD0010887
Expiry Date: 13.12.2012
Physical state at RT: Liquid
Colour: Brown
Active Components: 82.4% (17.6 % Water)
Density: 1.087 g/cm3
pH: 9.0 (1 % in water)
Boiling Point (C): 107 C
Stability: Stable
Storage Conditions: Room Temperature

Method

Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- 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 for experiment I (with and without metabolic activation):
5, 20, 100, 500, 1500, 3000, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
50, 75, 100, 125, 150, 175 µg/mL
Experiment I
without metabolic activation: 1.0, 2.5, 10.0 14.0, 16.0, 18.0, 20.0, 22.0 and 24.0 µg/mL
and with metabolic activation: 100, 120, 140, 180, 190, 210, 215, 225 and 235 µg/mL
Experiment II
without metabolic activation: 2.5, 10.0, 25.0, 50.0, 75.0, 100.0, 115.0 and 130.0 µg/mL
and with metabolic activation: 75.0, 100.0, 125.0, 175.0, 182.5, 190.0, 197.5, 205.0 and 212.5 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment)
The test item was dissolved in cell culture medium, processed by stirring for 10 min and diluted prior to treatment. All concentrations used referred to the active component of the test item (correction factor 1.214)

Controlsopen allclose all
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation

Migrated to IUCLID6: 300 µg/mL
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation

Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(Experiment I without S9: ≥ 18.0 μg/mL; experiment I with S9: ≥ 140 μg/mL; Experiment II without S9: ≥ 100.0 μg/mL; Experiment II with S9:≥ 182.5 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In a mammalian cell gene mutation assay (HPRT locus),V79cells culturedin vitrowere exposed toPentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine saltsdissolved incell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment)at concentrations of

-1.0, 2.5, 10.0 14.0, 16.0, 18.0, 20.0, 22.0 and 24.0 µg/mL(without metabolic activation, Experiment I)

-100, 120, 140, 180, 190, 210, 215, 225 and 235 µg/mL(with metabolic activation, Experiment I)

-2.5, 10.0, 25.0, 50.0, 75.0, 100.0, 115.0 and 130.0 µg/mL(without metabolic activation, Experiment II)

-75.0, 100.0, 125.0, 175.0, 182.5, 190.0, 197.5, 205.0 and 212.5 µg/mL(with metabolic activation, Experiment II).

Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine saltswas testedup to cytotoxic concentrations.

In experiment I without metabolic activation the relative growth was 24.8% for the highest concentration (24.0 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 235 µg/mL with a relative growth of 14.3%.In experiment II without metabolic activation the relative growth was 16.0% for the highest concentration (130.0 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 212.5 µg/mL with a relative growth of 17.2%..

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.70 was found at a concentration of 16.0 µg/mL with a relative growth of 90.7%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.51 was found at a concentration of 215 µg/mL with a relative growth of 21.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.07 was found at a concentration of 75.0 µg/mL with a relative growth of 78.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.03 was found at a concentration of 197.5 µg/mL with a relative growth of 31.4%.

The positive controlsdidinduce the appropriate response. 

There wasno evidence of a concentration related positive responseof induced mutant colonies over background.

This study is classified asacceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts is non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts is investigated for its mutagenic potential according to the OECD Guideline 476 (HPRT). No mutagenic effect was found.