Reaction mass of 1,3-diacetoxypropan-2-yl 12-acetoxyoctadecanoate and 2,3-diacetoxypropyl 12-acetoxyoctadecanoate

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:

Between 2001-10-18 and 2001-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP study with no deviations.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-530-8

EC Name:

-

Cas Number:

736150-63-3

Molecular formula:

Not applicable

IUPAC Name:

1,3-bis(acetyloxy)propan-2-yl 12-(carboxyoxy)octadecanoate; 2,3-bis(acetyloxy)propyl 12-(acetyloxy)octadecanoate

Details on test material:

Name of test material (as cited in study report): Emulsifier TS-ED 532
- Lot/batch No.: item 175540 batch 10102
- Expiration date of the lot/batch: 2002-04-26

Method

Target gene:

Strain Target mutation Mutation type
TA 1535 hisG46 Base-pair substitution
TA 100 hisG46 Base-pair substitution
TA 98 hisD3052 Frame shift
TA 102 hisG428 Frame shift
TA 1537 hisC3076 Base-pair substitution

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

NA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50, 160, 500, 1600 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)

Justification for choice of solvent/vehicle: solubility

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (100 ug/plate)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix: 1 ug/plate (TA100, TA1535)

Controls 2

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix: 1 ug/plate (TA98, TA1537)

Controls 3

Positive control substance:

other: cumene peroxide

Remarks:

Without S9-mix: 100 and 25 ug /plate (TA102)

Controls 4

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9-mix: 2 ug/plate (TA100, TA98, TA1535, TA1537) and 4 ug/plate (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method and the preincubation method.

DURATION
- Preincubation period: the plate incorporation and pre-incubation test was used, in which plates were incubated for approx. 72 hours.
- Exposure duration: approx 72 hours.
- Expression time (cells in growth medium): NA
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): NA

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: triplicate replications were used in the preliminary toxicity test and main tests.

NUMBER OF CELLS EVALUATED: NA

DETERMINATION OF CYTOTOXICITY
- Method: NA

OTHER EXAMINATIONS:
- Determination of polyploidy: NA
- Determination of endoreplication: NA
- Other: NA

Evaluation criteria:

Validity criteria:
- negative/positive control data were consistent with historical control data
- positive control showed marked increase over the concurrent negative control
- evaluation was not restricted by loss of plates (e.g. through contamination)

Mutagenic activity:
- dose-related increases in number of revertant colonies at one or more test points
- increases are reproducible between replicate plates
- increases are more than twice the corresponding negative control

Weak mutagenicity:
- dose-related increases in number of revertant colonies at one or more test points
- increases are reproducible between replicate plates
- increase are between 1.5 and 2 fold greater than the negative control

Statistics:

The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values using the Analysis of
Variance test. When this test showed statistical significanct differences in the data. Dunnett's test was used to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicate plates.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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
- Effects of pH: No information
- Effects of osmolality: No information
- Evaporation from medium: No information
- Water solubility: None
- Precipitation: None
- Other confounding effects: NA

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values were acceptable and compatible with the historical control values.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed at any concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 range of concentrations used in test 1 and 2.

Metabolic Activation	Test Substance Concentration (μg/mL)
Present
Test 1	50,160, 500, 1600, 5000
Test 2	50,160, 500, 1600, 5000
Absent
Test 1	50, 160, 500, 1600, 5000
Test 2	50, 160, 500, 1600, 5000

Table 2. Cytotoxic and genotoxic effects.

Metabolic Activation	Test Substance Concentration (µg/plate) Resulting in:
	Cytotoxicity in Preliminary Test	Cytotoxicity in Main Test	Precipitation	Genotoxic Effect
Present
Test 1	>5000	>5000	none	none
Test 2		>5000	none	none
Absent
Test 1	>5000	>5000	none	none
Test 2		>5000	none	none

Generally, TS-ED 532 was not toxic to bacteria, although it caused slight toxicity (small reductions in the numbers of revertant colonies) at the higher dose level in strains TA 100 and TA 1535 in a few cases. No biologically or statistically increases in the numbers of revertant colonies, compared to the negative control values, were observed in any tester strain after treatment with TS-ED 532 at any dose level, neither in the absence nor presence of S-9 mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

TS-ED 532 was non-mutagenic in the Ames test using S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 with and without metabolic activation.

Executive summary:

The mutagenicity of TS-ED 532 was tested in the Ames test using S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in accordance with OECD 471. The test was conducted in duplicate, once using the direct plate incorporation method and once using the preincubation method. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to TS-ED 532 at any concentration tested neither in the presence nor in the absence of S9 mix. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, confirming the activity of the S9 mix, and the sensitivity of the tester strains.

In conclusion, TS-ED 532 was found to be non-mutagenic in the Ames test using S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 with and without metabolic activation.