Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jan. 2017 to 27 Jan. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

EC Number:

270-471-7

EC Name:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Cas Number:

68441-67-8

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Test material form:

liquid

Details on test material:

Designation in Test Facility: 16102602G
Date of Receipt: 26. Oct. 2016
Condition at Receipt: Room temperature, in proper conditions

Specification
Name Hatcol 3178
Batch no. 2011280182
Appearance: Clear liquid
CAS No.: 68441-67-8
EINECS-No.: 270-471-7
Molecular formula: C33H60O8 to C41H76O8 (in CH2 unit increments)
Molecular weight: 692 daltons
Purity: 99.6 %
Homogeneity: homogeneous liquid
Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date: 18. Jul. 2011
Expiry date: 01. Nov. 2019
Storage: Room Temperature (20 ± 5°C)

Storage in Test Facility
The test item was stored in a closed flask at 15.8 – 22.0 °C, protected from humidity.

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 97a

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the experiment 1a and 1b: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
The following nominal concentrations were prepared for the experiment 2: 5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.

Vehicle / solvent:

Ethanol was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylenediamine; 2-Aminoanthracene

Details on test system and experimental conditions:

Culture of Bacteria
Eight hours before the start of each experiment, one vial of permanent culture from each strain was taken from the deep freezer, and an aliquot was put into a culture flask containing nutrient broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Conduct of Experiment
Preparations
In the days before each test (exact production dates are documented in the raw data), the media and solutions were prepared.
On the day of the test, the bacteria cultures were checked for growth. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Description of the Method
General preparation
Per strain and dose, three plates with and three plates without S9 mix were used.
Top agar base was melted in a microwave oven, after melting, 10 mL of histidine-biotin solution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 μL S9 mix or phosphate buffer (for test without metabolic activation)
-100 μL bacteria suspension
-2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation)
-100 μL bacteria suspension
After pre-incubation, 2000 μL overlay agar (top agar) was added, the tube was gently vortexed and the mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Rationale for test conditions:

In accordance with the test guidelines

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 97a

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

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:

Experiment 1a
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges (1 exception).
Solubility and Toxicity
In the experiment 1a, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.
To verify this result, a further experiment was performed.
Due to an experimental error, no evaluation of the bacteria strains TA97a and TA98 was possible.

Experiment 1b
Confirmation of the Criteria and Validity
Both strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the experiment 1b, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.
To verify this result, a further experiment was performed.

Experiment 2
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls (one exception) were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.

Acceptability of Study, Discussion
In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate.
In all three experiments, the test item caused no cytotoxicity towards all bacteria strains
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.
Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls, one exception) were within the range of the historical data of the test facility.
Nearly all numbers of revertant colonies of the positive controls (only one exception) were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:

The test item Hatcol 3178 showed no increase in the number of revertants in all bacteria strains in all three experiments.
Nearly all negative and 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.
Based on the results of this study it is concluded that Hatcol 3178 is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Executive summary:

Title of Study: Determination of the mutagenic potential of Hatcol 3178 with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14

 

Findings and Results:

Three valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

The test item Hatcol 3178 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

 

In experiment 1a, Hatcol 3178 (dissolved in ethanol) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

Hatcol 3178 showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item Hatcol 3178 showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Due to an experimental error, no evaluation of the bacteria strains TA97a and TA98 was possible.

 

In experiment 1b, Hatcol 3178 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA97a and TA98 using the plate incorporation method.

Hatcol 3178 showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item Hatcol 3178 showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

On the base of the experiments 1a and 1b, Hatcol 3178 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

Hatcol 3178 showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item Hatcol 3178 showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item Hatcol 3178 caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item Hatcol 3178 did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that Hatcol 3178 is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.