Isostearic acid, esters with methyl α-D-glucoside

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-07-15 to 2008-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

June 8, 2000

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isostearic acid, esters with methyl α-D-glucoside

EC Number:

700-680-5

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Isostearic acid, esters with methyl α-D-glucoside

Method

Target gene:

The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP2 uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and E. coli wP2 uvrA are capable of detecting base-pair substitution mutagens.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryphtophane auxotroph

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

S9-mix induced by combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw), rat liver micosomal enzymes prepared in house from adult male Wistar rats, which were obtained from Charles River, Sulzfeld Germany

Test concentrations with justification for top dose:

Dose-range finding test:
3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Main test: 33, 100, 333, 1000, 3330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Stability of test substance in vehicle: unknown
- Solubility in vehicle: not indicated

Controls

Controls 1

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, with S9-mix, further details see "any other information on materials and methods incl. tables"

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: for TA 98, 10 µg, without S9-mix, DMSO as solvent

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: for TA 100, 650 µg, without S9-mix, DMSO as solvent

Controls 4

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: for WP2uvrA, 10 µg, without S9-mix, DMSO as solvent

Controls 5

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: for TA 1537, 60 µg, without S9-mix, in Milli-Q water as solvent

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: for TA 1535, 5 µg without S9-mix, in physiological saline as solvent

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): Salmonella typhimurium stains: histidine; E. coli stain: tryptophane

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: reduction of the background lawn or reduction of the spontaneous reversion rate

DOSE RANGE FINDING TEST:
- selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix.- Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate.
- The highest concentration of test substance in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
- Precipitation of test substance on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards and at 3330 µg/plate and above at the end of the incubation period.
- Toxicity: to determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding crlteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed at the start of the incubation period at concentrations of 1000 and 3330 µg/plate and at the top dose of 3330 µg/plate at the end of the incubation period.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
Toxicity: no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: no increase in the number of revertants was observed upon treatment with test substance under all conditions tested.
COMPARISON WITH HISTORICAL CONTROL DATA:
In this study, the strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

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

Based on the results of this study it is concluded that Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to Isostearic acid, esters with methyl α-D-glucoside, at concentrations of 33, 100, 333, 1000, and 3330 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method). Isostearic acid, esters with methyl α-D-glucoside did not induce a significant dose-related increase in the number of revertant colonies in each of the five tester strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in an independently repeated experiment. No cytotoxic effects of the test substance were observed. Precipitation was observed at concentrations of 1000 and 3330 µg/plate. The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. There was no evidence of induced mutant colonies over background.     Based on the results of this study it is concluded that Isostearic acid, esters with methyl α-D-glucoside is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  

This study is classified as acceptable. It satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.