Reaction product of stearic acid and diglycerol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to the two in vitro studies available conducted on a structural analogue substance, both with a negative outcome, as conclusion ESTER B doses not induce genetic toxicity adverse effects.

Link to relevant study records

Reference

Reference 1

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

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

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.

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 / 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

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: [ ] [ ] [*]

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

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

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

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

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.

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: [ ] [ ] [*]

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

cytogenetic effects evaluations

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 with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

OECD 473 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:

other: in vitro mammalian chromosome aberration test (migrated information)

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.
The test substance was placed in a sealed container to avoid direct sunlight and store at room temperature until use. After completion of the test, residual test substances were analyzed at the source of the test substance
As a result, there was no problem in stability.

Every time of use the test substance was dissolved in DMSO dehydrated using molecular sieve to prepare a stock solution for preparation. The stock solution to be prepared is gradually washed with a solvent
After the dilution, the treatment was promptly carried out.

Target gene:

Chinese hamster lung-derived fibroblast cell line(CHL / IU) was chosen.
On November 15, 1984, received dispensation from the National Institute of Hygiene Experiment (currently the National Institute of Food Hygiene of Japan), and added dimethylsulfoxide (DMSO, Merck).

Cells were cultured under conditions of CO 2 concentration of 5% and 37 ° C. using a CO 2 incubator (SANYO Electric Biomedica).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

On November 15, 1984, received dispensation from the National Institute of Hygiene Experiment (currently the National Institute of Food Hygiene of Japan), and added dimethylsulfoxide (DMSO, Merck).
Was added at 10 vol% and then stored in liquid nitrogen. For the test, frozen cells were thawed and used passaged every 3 to 5 days. In the cell proliferation inhibition test
Cells with passage number 9, chromosome aberration test in passage number 17, and chromosome aberration test (additional test) used cells with passage number 24

PREPARATION OF CULTURE SOLUTION:
After inactivation (56 ° C., 30 minutes) calf serum (Invitrogen) was added to Eagle-MEM liquid medium (Asahi Technoglass) to a final concentration of 10 vol%, it was used for the test.
The culture solution after preparation was stored in a cool dark place (4 ° C.).

Cells were cultured under conditions of CO 2 concentration of 5% and 37 ° C. using a CO 2 incubator (SANYO Electric Biomedica).

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:

Cells were cultured under conditions of CO 2 concentration of 5% and 37 ° C. using a CO 2 incubator (SANYO Electric Biomedica).

-S9 mix (short-term treatment); 0, 891, 1783, 3565 μg/mL
+S9 mix (short-term treatment); 0, 891, 1783, 3565 μg/mL
-S9 mix (continuous treatment); 0, 55.7, 111, 223 μg/mL

Vehicle / solvent:

Solvent : Dimethyl sulfoxide
Positive controls:
-S9 mix; Mitomycin C
+S9 mix; Cyclophosphamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

On cytogenetic effects of 9-cis-octadecenoic acid (2,3-dihydroxypropyl) ester on cultured cells, Chinese hamster cultured cells
(CHL / IU) was used to perform a chromosomal aberration test.

Based on the results of the cell proliferation inhibition test, 5 doses were administered in the concentration range of 223 to 3565 μg / mL with the maximum treatment concentration of 3565 μg / mL in the absence of the short-time treatment method S9 mix, In the presence of S9 mix, four doses were set in the concentration range of 446 to 3565 μg / mL with the maximum treatment concentration of 3565 μg / mL. Treatment in the presence and absence of S9 mix for 6 hours (Recovery time of 18 hours), specimens were prepared and examined for chromosome aberration induction by microscopic examination. In the absence and presence of S9 mix 891, 1783, 3565 μg / mL- Microscopic observation was carried out for 3 doses (common ratio 2).

As a result, no clear chromosomal abnormality was induced in either the absence or presence of S9 mix. Therefore, continuous treatment method by 24-hour treatment test was conducted. Seven doses were set in the concentration range of 55.7 to 3565 μg / mL with the maximum treatment concentration of 3565 μg / mL. 24 hours continuous treatment in the absence of S9 mix.
After that, specimens were prepared, and microscopic observation was conducted for three doses (common ratio 2) of 55.7, 111, and 223 μg / mL. Even in this treatment method, clear chromosomal abnormalities were not induced

Evaluation criteria:

One hundred per plate, that is, 200 metaphase images per dose, was observed under the microscope, and gaps as chromatographic morphological changes, chromatid cleavage
(ctb), chromosome breakage (csb), chromosome exchange (cte), chromosome exchange (cse) and other (oth) structural abnormalities. At the same time, the incidence of ploidy cells was recorded.
Chromosome analysis was performed according to the classification method 2) by the Japan Environmental Mutagen Society and Mammal Test Subcommittee.

The specimens were coded and chromosome analysis was performed.

Statistics:

Frequency of occurrence of cells with structural abnormality in each test group or ploidy cells was tested using Fisher's direct probability calculation method (significance level one side 2.5%). Also dose dependency was tested using Cochran Armitage trend test (significance level one side 2.5%). Significant difference was observed in the test substance treated group compared with the solvent control group It was judged to be positive when weak, and reproducibility or dose dependency was observed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

PRELIMINARY TEST INFO: As a result, in the continuous treatment method 24 hours treatment, the relative cell proliferation rate was below 50% at the highest dose of 3565 μg / mL and the concentration at which 50% inhibition of cell proliferation was 2738 μg / mL
met. In the short-time treatment method-S9 treatment and + S9 treatment, no definite cell proliferation inhibitory action was observed and a 50% cell proliferation inhibitory concentration could not be calculated (see results in Fig. 1 and 2 of the attachement as background material).


OVERALL RESULTS: The test results by the short-time treatment method are shown in Tables 1 and 2 of the attachemnt as background material. Chromosomal aberration test by continuous treatment method 24 hours treatment was carried out because it was judged as negative in the short time treatment method, and the results are shown in Table 3 attached in the document of background material.
Chromosome structure abnormality frequency in the group treated with senoic acid (2,3-dihydroxypropyl) ester was 0.0% at 55.7 μg / mL, 1.0% at 111 μg / mL and 0.0% at 223 μg / mL, And it was equivalent to the solvent control group. The induction tendency of ploidy cells was not observed at any dose. In addition, the cell proliferation inhibitory effect can be determined at any dose. Even though it was, it was not observed. On the other hand, marked induction of chromosome structural abnormality was observed in the cells treated with the positive control substance MMC.

Detailed test results by the short-time treatment method are shown in Tables 1 -3 of the attachemnt as background material.

No increase in chromosomal aberrations was observed with either the short-term treatment (-S9 mix and +S9 mix) or the continuous treatment

Genetic effects :

clastogenicity polyploidy

+ ? - + ? -

Without metabolic activation: [ ] [ ] [*] [ ] [ ] [*]

With metabolic activation: [ ] [ ] [*] [ ] [ ] [*]

Conclusions:

Negative

Executive summary:

The induction tendency of ploidy cells was not observed at any dose. In addition, the cell proliferation inhibitory effect can be determined at any dose. Even though it was, it was not observed.

Based on the above test results, it was confirmed that under this test condition, the substance, for Chinese hamster cultured cells, was judged as negative for chromosome aberrant induction.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the two in vitro studies available conducted on a structural analoge substance, both with a negative outcome, as conclusion ESTER B doses not induce genetic toxicity adverse effects.

Ester B is classified as no mutagenic according CLP Regulation.