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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Apr 2021 to 26 Apr 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guideline 471. Genetic Toxicology: Bacterial Reverse Mutation Test. (Adopted 21 July 1997, Corrected 26 June 2020).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
EC Number:
270-470-1
EC Name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Cas Number:
68441-66-7
Molecular formula:
not available due to complexity of the substance
IUPAC Name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Test material form:
liquid
Details on test material:
Identification: Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Batch (Lot) Number: 2019194336
Expiry date: 03 June 2022
Physical Description: Clear light yellow liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature

Additional information
Test Facility test item number: 212207/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Information about the purity and composition of the test item is not available since the test item is an UVCB (Substance of Unknown or Variable composition, Complex Reaction Products or Biological Materials). Since a sample relevant for the purpose of this study was tested, it was concluded that the study integrity was not affected by the omission of this
information.
Specific details on test material used for the study:
No further details specified in the study report.

Method

Target gene:
histidine and tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Test System: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g. OECD, EC).
Source: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]
The Salmonella typhimurium strains were checked at least every year to confirm their
histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100),
UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using
Tris-EDTA treatment (Ref.1). The strain was checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.
Stock cultures of the five strains were stored in the ultra-low freezer set to maintain -150°C.
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-Fraction
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
Test concentrations with justification for top dose:
Dose-range Finding Test: Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate.
In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate.
The highest concentration of the test item used in the subsequent mutation assays was 5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
Vehicle / solvent:
The test item formed a clear colourless solution in ethanol.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-AA
Details on test system and experimental conditions:
Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 36.6 - 39.3°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Experimental Design
Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assays was
5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

First Experiment: Direct Plate Assay
The above mentioned dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(109 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in ethanol or control solution and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Second Experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were
pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays),
0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test item in ethanol or control solution. After the pre-incubation period 3 mL molten top agar was added to the solutions. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Rationale for test conditions:
In accordance with test guidelines
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
First Experiment: Direct Plate Assay
The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and
5000 μg/plate.

Precipitate
Precipitation of the test item on the plates was observed at concentrations of 1600 µg/plate and upwards in tester strains TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 5000 μg/plate in tester strains TA1535, TA1537 and TA98.

Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
In strain TA100 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

Mutagenicity
In the direct plate test, no biologically relevant increase in the number of revertants was observed upon treatment with the test item under all conditions tested. In tester strains WP2uvrA (absence of S9-mix) and TA1537 (presence of S9-mix), the test item induced 2.1-fold and 4.0-fold increases in the number of revertant colonies compared to the solvent control, respectively. These increases were not dose related and well within the historical database. Therefore the observed increases were not considered biologically relevant.

Second Experiment: Pre-Incubation Assay
To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitate
The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards in the absence of S9-mix and at the top dose level of 5000 µg/plate in the presence of S9-mix.

Toxicity
There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In strain TA100 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reduction are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

Mutagenicity
In the pre-incubation test, no biologically relevant increase in the number of revertants was observed upon treatment with the test item under all conditions tested. In tester strain TA1537 (absence of S9-mix), the test item induced a 5.0-fold increase in the number of revertant colonies compared to the solvent control. The increase was not dose related, well within the historical database and linked to a low solvent control. Therefore the observed increase was not considered biologically relevant.

Formulation Analysis
Accuracy
In the vehicle, no test item was detected.
The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

Homogeneity
The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability
Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%, for the low samples. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours. For the high samples, the relative difference was > 10% (i.e. -12%, see deviation).

Any other information on results incl. tables

 Dose-Range Finding Test: Mutagenic Response of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 

Direct Plate Assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain

 

TA100

WP2uvrA

 

 

 

 

Without S9-mix

Positive control

882

±

54

 

1484

±

44

 

 

 

 

 

Solvent control

72

±

10

 

10

±

3

 

 

 

 

 

1.7

84

±

10

 

14

±

2

 

 

 

 

 

5.4

79

±

7

 

12

±

0

 

 

 

 

 

17

74

±

16

 

16

±

4

 

 

 

 

 

52

79

±

11

 

10

±

2

 

 

 

 

 

164

73

±

10

 

15

±

1

 

 

 

 

 

512

62

±

14

NP

15

±

1

NP

 

 

 

 

1600

76

±

14

SP

21

±

1

SP

 

 

 

 

5000

84

±

3

nSP

16

±

4

nSP

 

 

 

 

With S9-mix

Positive control

718

±

97

 

360

±

14

 

 

 

 

 

Solvent control

57

±

13

 

11

±

2

 

 

 

 

 

1.7

51

±

3

 

16

±

4

 

 

 

 

 

5.4

58

±

23

 

18

±

5

 

 

 

 

 

17

58

±

11

 

20

±

6

 

 

 

 

 

52

54

±

3

 

19

±

5

 

 

 

 

 

164

46

±

11

 

15

±

8

 

 

 

 

 

512

55

±

7

NP

16

±

2

NP

 

 

 

 

1600

63

±

8

SP

16

±

4

SP

 

 

 

 

5000

56

±

8

nSP

20

±

9

nSP

 

 

 

 

NP = No precipitate

SP = Slight precipitate

n = Normal bacterial background lawn

 

Experiment 1: Mutagenic Response of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 

Direct Plate Assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium

 

TA1535

TA1537

TA98

Without S9-mix

Positive control

910

±

57

 

1092

±

158

 

1498

±

359

 

Solvent control

9

±

4

 

7

±

4

 

19

±

1

 

17

9

±

5

 

4

±

0

 

21

±

6

 

52

7

±

0

 

5

±

2

 

17

±

2

 

164

7

±

7

 

7

±

2

 

13

±

2

 

512

11

±

4

 

5

±

2

 

20

±

2

 

1600

6

±

3

NP

9

±

5

NP

18

±

3

NP

5000

13

±

4

nSP

4

±

2

nSP

17

±

6

nSP

With S9-mix

Positive control

229

±

27

 

241

±

14

 

1209

±

101

 

Solvent control

10

±

1

 

2

±

2

 

24

±

10

 

17

10

±

3

 

3

±

3

 

25

±

8

 

52

9

±

3

 

5

±

2

 

19

±

2

 

164

9

±

6

 

5

±

2

 

22

±

8

 

512

11

±

4

 

8

±

4

 

23

±

8

 

1600

12

±

1

NP

8

±

5

NP

22

±

9

NP

5000

14

±

9

nSP

4

±

2

nSP

26

±

2

nMP

MP = Moderate Precipitate

NP = No precipitate

SP = Slight precipitate

n = Normal bacterial background lawn

 

Experiment 2: Mutagenic Response of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 

Pre-incubation Assay

Dose

(µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.

 

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

967

±

185

 

56

±

11

 

1590

±

84

 

782

±

56

 

839

±

129

 

Solvent control

7

±

0

 

1

±

1

 

14

±

10

 

107

±

10

 

21

±

6

 

17

10

±

6

 

3

±

2

 

17

±

7

 

107

±

12

 

23

±

4

 

52

11

±

1

 

3

±

2

 

17

±

5

 

100

±

13

 

18

±

1

 

164

9

±

4

 

3

±

2

 

15

±

4

 

103

±

8

 

20

±

1

 

512

9

±

2

NP

3

±

2

NP

17

±

10

NP

110

±

19

NP

22

±

8

NP

1600

11

±

1

SP

5

±

2

SP

14

±

3

SP

122

±

20

SP

24

±

9

SP

5000

11

±

3

nSP

2

±

1

nMP

14

±

2

nSP

118

±

9

nSP

19

±

8

nMP

With S9-mix

Positive control

112

±

8

 

33

±

7

 

367

±

26

 

636

±

151

 

345

±

24

 

Solvent control

12

±

4

 

6

±

2

 

23

±

1

 

52

±

11

 

24

±

10

 

17

9

±

2

 

4

±

1

 

20

±

3

 

57

±

10

 

27

±

5

 

52

8

±

5

 

4

±

1

 

27

±

4

 

48

±

3

 

27

±

4

 

164

7

±

2

 

2

±

2

 

24

±

1

 

43

±

8

 

14

±

2

 

512

11

±

4

 

5

±

2

 

19

±

4

 

46

±

7

 

32

±

2

 

1600

11

±

1

NP

2

±

2

NP

18

±

2

NP

47

±

11

NP

21

±

2

NP

5000

16

±

3

nSP

2

±

1

nMP

24

±

1

nSP

57

±

13

nSP

27

±

7

nSP

MP = Moderate Precipitate

NP = No precipitate

SP = Slight precipitate

n = Normal bacterial background lawn

 

Historical Control data of the Solvent Control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 26

4 – 25

2 – 24

2 – 17

4 – 61

6 – 60

58 – 188

50 – 176

9 – 61

11 – 68

Mean

9

10

5

5

14

18

109

101

24

29

SD

3

3

2

2

5

6

19

21

9

10

Total number of plates

2504

2484

2528

2493

2523

2528

2566

2508

2385

2361

SD = Standard deviation

Historical control data from experiments performed between Nov 2017 and Nov 2020.

 

Historical Control Data of the Positive Control Items

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

107 – 1530

78 – 1481

64 – 1475

52 – 1843

379 – 2118

265 – 2077

452 – 1993

397 – 2666

93 – 1999

109 – 1968

Mean

972

289

818

293

1252

945

865

1432

1196

421

SD

170

108

370

142

251

378

181

398

526

186

Total number of plates

2360

2365

1979

2384

2451

2379

2382

2387

2269

2267

SD = Standard deviation

Historical control data from experiments performed between Nov 2017 and Nov 2020.

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S.typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

 

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

 

Batch 2019194336 of the test item was a clear light yellow liquid. The vehicle of the test item was ethanol.

 

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%). The low dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours. For the high samples, the relative difference was > 10% (i.e. -12%, seedeviation).

 

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

 

In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards in the absence of S9-mix and at the top dose level of 5000 µg/plate in the presence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. 

 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

The test item did not induce a biologically relevant, dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

In conclusion, based on the results of this study it is concluded that Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.