Octanoic acid, ester with 1,2,3-propanetriol

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:

17 Apr -21 Apr 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions. Only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1983

Deviations:

yes

Remarks:

only 4 S. typhimurium strains used instead of 5 as required according to the current criteria. Only one concentration (5000 µg/plate) tested due to insolubility of the test substance.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Glycerol tristearate

EC Number:

209-097-6

EC Name:

Glycerol tristearate

Cas Number:

555-43-1

Molecular formula:

C57H110O6

IUPAC Name:

propane-1,2,3-triyl trioctadecanoate

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/β-Naphthoflavone

Test concentrations with justification for top dose:

5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 95% ethanol (100 and 50 µL/plate in the plate incorporation and preincubation tests, respectively).
- Justification for choice of solvent/vehicle: in a preliminary test, the solubility of the test compound was determined in a number of solvents suitable for the Ames test, namely water, dimethylsulfoxide, glycerol, dimethyl formamide, formamide, ethanol, acetone, dioxane, tetrahydrofuran and tetrahydrofurfuryl alcohol. In all these solvents, the test compound could not be dissolved in appreciable amounts. Therefore, a suspension of the test material was prepared on the day of testing by mixing the test compound with 95% ethanol, stirring it on a magnetic stirrer and using samples of this stirred suspension for testing. No stability testing or composition analysis was performed on the dosing suspension.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-nitrofluorene (2-NF; 2.5 µg/plate for TA98); sodium azide (SA; 5 and 2.5 µg/plate for TA100 and TA1535, respectively); 9-aminoacridine (9-AA; 40 µg/plate for TA1537; +S9: 2-aminoanthracene (2-AA; 2.5 µg/plate for all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: preincubation

DURATION
- Preincubation period: 30 min (second experiment)
- Exposure duration: 72 h (first and second experiment)

NUMBER OF REPLICATIONS: 3 replications in the first and second experiment, respectively.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

Evaluation criteria:

Criteria for determination of a valid test:

The following criteria had to be met for the mutagenicity assay to be considered valid:
- In the solvent control, each tester strain culture exhibited a characteristic mean number of spontaneous revertants.
- To ensure that appropriate numbers of bacteria were plated, overnight culture titers had to be in excess of 1E08 bacteria/mL.
- The mean of each positive control exhibited a significant increase in the number of revertants over the mean value of the respective vehicle control.
- In a standard Ames test, at least four non-toxic dose levels were required to evaluate the assay data. In the current test, due to non-solubility of the test compound, only a limit dose was employed.

Criteria for evaluation of test results:

For a test compound to be considered positive, it had to (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. In a standard Ames test, this increase had to be accompanied by a dose response towards increasing concentrations of the test article. In the current test, a reproducible caused by a treatment with the limit dose would have been sufficient. A test article that did not meet these criteria would be called non-mutagenic in bacteria. Single increases in revertant frequencies, which were not reproducible in two independent tests were considered non-relevant.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

due to insolubility, the test mateial was tested as suspension at a single limit concentration of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance is not soluble in water.
- Precipitation: due to non-solubility of the test susbtance in all solvents used routinely in the Ames test, the tester strains were exposed to a suspension of the test compound at a single limit concentration of 5000 µg/plate. Test compound-treated bacterial colonies were counted by hand, as the precipitate formed by the test compound prevented automatic counting.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test Results of Experiment 1 (plate incorporation).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type		Frameshift type
		TA 100	TA1535	TA98	TA1537
–	untreated	110	8	31	12
–	Ethanol	105	7	33	16
–	5000	125P	3P	30P	20P
Positive controls, –S9	Name	SA	SA	2-NF	9-AA
	Concentrations (μg/plate)	5	2.5	2.5	40
	Mean No. of colonies/plate (average of 3)	575	267	123	125
+	untreated	126	11	25	12
+	Ethanol	116	14	33	16
+	5000	136P	8P	29P	10P
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5
	Mean No. of colonies/plate (average of 3)	1394	123	835	120

 

SA = sodium azide

2-NF = 2-nitrofluorene

9-AA = 9-aminoacridine

2-AA = 2-Aminoanthracene

P = Precipitate

 

 

Table 2. Test Results of Experiment 2 (preincubation).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type		Frameshift type
		TA 100	TA1535	TA98	TA1537
–	untreated	142	13	30	14
–	Ethanol	126	9	28	11
–	5000	127P	8P	24P	12P
Positive controls, –S9	Name	SA	SA	2-NF	9-AA
	Concentrations (μg/plate)	5	2.5	2.5	40
	Mean No. of colonies/plate (average of 3)	575	267	123	125
+	untreated	134	7	26	13
+	Ethanol	155	11	27	10
+	5000	128P	10P	31P	13P
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5
	Mean No. of colonies/plate (average of 3)	839	126	657	76

 

SA = sodium azide

2-NF = 2-nitrofluorene

9-AA = 9-aminoacridine

2-AA = 2-Aminoanthracene

P = Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative