Reaction product of stearic acid and diglycerol

Genetic toxicity: in vitro

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Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

OECD 471 study performed on a structural analogue of the target substance
see detailes in the attached justification for read-across

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

at room temperature appears as a yellow waxy paste

Specific details on test material used for the study:

The 9-cis-octadecenoic acid (2,3-dihydroxypropyl) ester (lot number: 404230) is the test substance, 99.93% pure (0,05% of tocopherol extracted as impurity and containing 0.02% of phosphoric acid).
The test subastance has the form of a pale yellow wax. It is insoluble in water, but is readily soluble in DMSO.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Every time the test substance was dissolved using DMSO dehydrated with molecular sieve, it was used as a preparation stock solution. Preparation stock solution is used sequentially with solvent to be used.
After diluting to constant concentration, treatment was promptly carried out.

Method

Target gene:

Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA

Salmonella typhimurium was deposited from Professor Ames BN of the University of California on September 9, 1988, and on March 16, 1988 for Escherichia coli, the National Institutes of Health
Chemical Food Hygiene Laboratory). Characteristic tests of strains were carried out from September 16 to September 19, 2003, and the strains used for the test retain prescribed characteristics
I confirmed that.

After addition of dimethylsulfoxide (DMSO, Merck), 0.2 mL aliquots of each bacterial suspension were dispensed into a cryopreservation tube. It was frozen using liquid nitrogen
And stored at -80 ° C. in an ultra-low temperature freezer.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix received phenobarbital + 5,6-benzoflavone from liver of male Sprague-Dawley rats. Ingredient S9mix 1 mL: S9 0.3 mL; MgCl2 5 μmol/0.1 mL; KCl 33 μmol/0.1 mL; G6P 5 μmol/0.1 mL; NADP 4 μmol/0.1 mL; HEPES pH 7.2 4 μmol/0.2 mL; Distilled water 0.1 mL

Test concentrations with justification for top dose:

[Dose-finding study]
-S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000μg/plate (all strains)
+S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000μg/plate (all strains)
[Dose-finding restudy]
-S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000μg/plate (all strains)
+S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000μg/plate (all strains)
[Dose-finding additional study]
-S9 mix; 0, 6.25, 12.5, 25.0, 50.0, 100, 200 μg/plate
(TA100), 0, 3.13, 6.25, 12.5, 25.0, 50.0, 100 μg/plate
(TA98), 0, 0.0977, 0.195, 0.391, 0.781, 1.56, 3.13, 6.25,12.5 μg/plate (TA1537)
+S9 mix; 0, 6.25, 12.5, 25.0, 50.0, 100, 200 μg/plate (TA1537)
[Main study]
-S9 mix; 0, 4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA100, TA98),
0, 156, 313, 625, 1250, 2500, 5000μg/plate (TA1535, WP2 uvrA),
0, 0.153, 0.305, 0.610, 1.22, 2.44, 4.88, 9.77 μg/ plate (TA1537)
+S9 mix; 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA100),
0, 156, 313, 625, 1250, 2500, 5000 μg/plate (TA1535, WP2 uvrA, TA98),
0, 9.77, 19.5, 39.1,78.1, 156, 313 μg/plate (TA1537)

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) used as solvent

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO as negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: (2-AA) 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
In accordance with the pre-incubation method (which is an improvement method of Ames et al. 'S original method 1), tests were conducted for each group without addition of S9 mix and addition group. For test tubes, use 100 μL of the solvent for test, the test substance solution or the positive control substance solution, then 500 μL of 0.1 mol / L sodium phosphate buffer solution (pH 7.4) in the case of the group without S9 mix, 500 μL of S9.
In the case of the mix addition group, 500 μL of S 9 mix was added, 100 μL of the test bacterial solution was further added, and shake culture (preincubation) was carried out at 37 ° C. for 20 minutes. After completion of culture, beforehand 2 mL top agar kept at 45 ° C was added and the mixture was layered on the plate. After culturing each plate for 48 hours at 37 ° C., the test strain was tested against the test strain of the test substance.
In order to confirm the inhibitory effect on growth, the growth condition of the test strain on the plate was observed using a stereoscopic microscope (× 40). Colonies generated by reversion were then measured. For the measurement, a colony analyzer (CA-11, system science) was used. In addition, due to growth inhibition, use of colony analyzer is inappropriate.
In case, counted visually. Three plates were used for each concentration.

Statistics:

Genetic effects:

Salmonella typhimurium TA100, TA1535, TA98, TA1537
+ ? -
Without metabolic activation: [ ] [ ] [*]
With metabolic activation: [ ] [ ] [*]

Escherichia coli WP2 uvrA
+ ? -
Without metabolic activation: [ ] [ ] [*]
With metabolic activation: [ ] [ ] [*]

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the strain, reversion mutant colonies at least twice the solvent control group were induced. When treated with the test substance, both S9 mix-free group and added group had a dose of 313 μg / plate or more.
Precipitates of white powder (partly lumpy in S9 mix-absent group) were observed in dose. At the time of colony counting, a dose of 1250 μg / plate or more in S9 mix-free group and S9
Precipitates of white powders (some lumps in S9 mix-absent group) were observed at doses of 625 μg / plate or more in the mixture-added group. Depending on the influence of the precipitate, S9 mix non-additive group.
For both the supplement group and the addition group, it was judged that the use of the colony analyzer was inappropriate at the dose of 1250 μg / plate or more, and colonies were counted visually.

Any other information on results incl. tables

The results of the test are shown in Tables 7 and 8 of the attached document for background material.

Salmonella typhimurium TA100, TA1535, TA98, TA1537

                 +  ?  -

Without metabolic activation: [ ] [ ] [*]

With metabolic activation: [ ] [ ] [*]

Escherichia coli WP2 uvrA

                  +  ?  -

Without metabolic activation: [ ] [ ] [*]

With metabolic activation: [ ] [ ] [*]

Applicant's summary and conclusion

Conclusions:

Negative

Executive summary:

No increase in revertant colonies was observed in the test with either the non-activation method (-S9 mix) or the activation method (+S9 mix).

The gene mutation assay for the test substance it was determined to be negative.