Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity - in vitro

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.

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

histidine and tryptophan

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.

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.

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).

 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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Mar 2021 to 23 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

OECD Guideline 473. In Vitro Mammalian Chromosome Aberration Test (adopted 29 July 2016).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.10: "Mutagenicity - In Vitro Mammalian Chromosome Aberration Test". Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

polyploid cells and cells with endoreduplicated chromosomes

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (OECD, EC).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to
35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2020) are presented below:
Dose-range finding study: age 30, AGT = 15.7 h
First cytogenetic assay: age 25, AGT = 13.1 h
Second cytogenetic assay: age 29, AGT = 14.6 h

Cell Culture
Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies).
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was determined by the solubility (3 h exposure time and 24 h exposure time).
First Cytogenetic Assay: Without and with S9-mix: 78, 156 and 313 µg/mL culture medium (3 h exposure time, 24 h fixation time).
Second Cytogenetic Assay:
Without S9-mix:10, 100, 200, 300, 400, 500 and 625 µg/mL culture medium (24 h exposure time, 24 h fixation time).
Without S9-mix: 10, 200 and 400 µg/mL culture medium (24 h exposure time, 24 h fixation time).

Vehicle / solvent:

The vehicle for the test item was ethanol (Extra pure, Merck, Darmstadt, Germany).

Solvent for Positive Controls
Hanks’ Balanced Salt Solution (HBSS) (Life technologies, Bleiswijk, The Netherlands), without calcium and magnesium.
All reference stock solutions were stored in aliquots in the freezer set to maintain -20°C in the dark. These solutions were thawed immediately before use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Dose-range Finding Test
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. the test item was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 h and 24 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.
The highest tested concentration was determined by the solubility of the test item in the culture medium.
After 3 h exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h fixation time).
Cytotoxicity of the test item in the lymphocyte cultures was determined using the mitotic index.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was determined by the solubility (3 h exposure time and 24 h exposure time).

Cytogenetic Assay
The cytogenetic assay was carried out as described by Evans, 1984 with minor modifications. the test item was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.

First cytogenetic assay
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of the test item for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.
Based on the mitotic index of the dose-range finding test and the first cytogenetic assay and on the solubility of the test item in the culture medium appropriate dose levels were selected for the second cytogenetic assay. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. The follow up experiment was performed with the following modifications of experimental conditions.

Second cytogenetic assay
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of the test item for 24 h in the absence of S9-mix.
The cells were not rinsed after exposure but were fixed immediately after 24 h (24 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome Preparation
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of Slides
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Mitotic Index/Dose Selection for Scoring of the Cytogenetic Assay
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. The test item was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium.

Analysis of Slides for Chromosome Aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

Graphpad Prism version 8.4 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test
At concentrations of 313 µg/mL and 625 µg/mL and above the test item precipitated in the culture medium, at a 3 h and 24 h exposure time, respectively. Therefore, these concentrations were used as the highest concentrations of the test item. The pH and osmolarity of a concentration of 156 µg/mL were 7.710 and 373 mOsm/kg respectively (compared to 7.754 and 380 mOsm/kg in the solvent control). A concentration of 156 µg/mL was the highest non-precipitating concentration after 3 hours exposure.
In the dose-range finding test blood cultures were treated with 10, 20, 39, 78, 156 and 313 µg test item/mL culture medium with and without S9-mix at a 3 h exposure time. For the 24 h exposure time the blood cultures were treated with 20, 39, 78, 156, 313 and 625 µg test item/mL culture medium without S9-mix.

First Cytogenetic Assay
Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 78, 156 and 313 µg/mL culture medium (3 h exposure time, 24 h fixation time).
Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Both in the absence and presence of S9-mix, the test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.
Second Cytogenetic Assay
To obtain more information about the possible clastogenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to the test item in the absence of S9-mix for 24 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 10, 100, 200, 300, 400, 500 and 625 µg/mL culture medium (24 h exposure time, 24 h fixation time).
Further investigation showed that a concentration of 400 µg/mL already precipitated in the culture medium at a 24 h exposure time.
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 10, 200 and 400 µg/mL culture medium (24 h exposure time, 24 h fixation time).
The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Mitotic Index of Human Lymphocyte Cultures Treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Dose-Range Finding Test

Test item                                                         Number of metaphases

concentration (µg/mL)                                    Absolute       Number               Percentage
                                                                                                  of cells scored    of control

Without metabolic activation (-S9-mix)

3 h exposure time, 24 h fixation time

Control^a)		65		1000			100
10		67		1000			103
20		62		1000			95
39		53		1004			82
78		51		1006			78
156		64		1000			98
313	^b)	61		1001			94

 

24 h exposure time, 24 h fixation time

 

Control^a)		82		1002			100
20		66		1000			80
39		68		1000			83
78		57		1000			70
156		42		1000			51
313		39		1005			48
625	^b)	29		1000			35

 

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control^a)		58		1006			100
10		51		1001			88
20		54		1000			93
39		59		1004			102
78		51		1001			88
156		48		1000			83
313	^b)	50		1000			86

^a)   Ethanol.

^b)   The test item precipitated in the culture medium.

 

Mitotic Index of Human Lymphocyte Cultures Treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the First Cytogenetic Assay

Test item                                                      Number of metaphases^a)

concentration (µg/mL)                               Absolute           Number             Percentage
                                                                                                  of cells scored  of control

Without metabolic activation (-S9-mix)

3 h exposure time, 24 h fixation time

Control^b)		130	-	114		1000	-	1010		100
78		114	-	123		1004	-	1000		97
156		108	-	114		1010	-	1008		91
313	^c)	88	-	94		1000	-	1000		75
MMC-C; 0.5 µg/mL		71	-	66		1001	-	1000		56
MMC-C; 0.75 µg/mL		60	-	56		1004	-	1007		48

 

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control^b)		128	-	124		1000	-	1002		100
78		88	-	95		1000	-	1000		73
156		87	-	88		1002	-	1000		69
313	^c)	76	-	82		1000	-	1000		63
CP; 10 µg/mL		89	-	91		1005	-	1001		71

^a)   Duplicate cultures.

^b)    Ethanol.

^c)   The test item precipitated in the culture medium.

 

Chromosome Aberrations in Human Lymphocyte Cultures Treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Absence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time)

Conc	Ethanol (0.5% v/v)				78 µg/mL				156 µg/mL				313 µg/mL				MMC-C 0.5 µg/mL
Culture	A    B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				97				91				75				56
No. of Cells scored	150  150300				150  150300				150  150300				150  150300				75    150225
No. of Cells with aberrations (+ gaps) a)	0	1		1	0	1		1	0	1		1	0	1		1	38	33		****^) 71
No. of Cells with aberrations (- gaps)	0	1		1	0	1		1	0	1		1	0	1		1	38	33		****^) 71
g’
g”
b’		1				1				1				1			27	19
b”																	13	11
m’
m”
exch.																	15	9
dic																	2
d’
misc.
total aberr (+ gaps)	0	1			0	1			0	1			0	1			57	39
total aberr (- gaps)	0	1			0	1			0	1			0	1			57	39

^a)    Abbreviations used for various types of aberrations are listed inAppendix 2.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)    Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001, **** P < 0.0001.

 

Chromosome Aberrations in Human Lymphocyte Cultures Treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Presence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time)

Conc	Ethanol (0.5% v/v)				78 µg/mL				156 µg/mL				313 µg/mL				CP 10 µg/mL
Culture	A    B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				73				69				63				71
No. of Cells scored	150  150300				150  150300				150  150300				150  150300				150  150300
No. of Cells with aberrations (+ gaps) a)	1	2		3	0	0		0	0	1		1	0	0		0	32	27		****^) 59
No. of Cells with aberrations (- gaps)	1	2		3	0	0		0	0	1		1	0	0		0	32	27		****^) 59
g’
g”
b’										1							23	25
b”	1	2															8	5
m’
m”
exch.																	4	3
dic
d’
misc.																	1 endo
total aberr (+ gaps)	1	2			0	0			0	1			0	0			35	33
total aberr (- gaps)	1	2			0	0			0	1			0	0			35	33

^a)    Abbreviations used for various types of aberrations are listed inAppendix 2.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
The numerical variation endoreduplication (endo) was not counted as an aberration.

*)    Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001, **** P < 0.0001.

 

Mitotic Index of Human Lymphocyte Cultures Treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Second Cytogenetic Assay

Test item                                                      Number of metaphases^a)

concentration (µg/mL)                               Absolute           Number             Percentage
                                                                                                  of cells scored  of control

Without metabolic activation (-S9-mix)

24 h exposure time, 24 h fixation time

Control^b)		110	-	111		1000	-	1000		100
10		101	-	100		1001	-	1000		91
100		98	-	96		1000	-	1000		88
200		85	-	89		1000	-	1004		79
300		77	-	84		1010	-	1000		73
400	^c)	76	-	72		1000	-	1042		67
500	^c)	65	-	70		1000	-	1003		61
625	^c)	63		71		1000		1000		61
MMC-C; 0.2 µg/mL		46	-	51		1000	-	1001		44
MMC-C; 0.3 µg/mL		24	-	22		1000	-	1010		21

^a)   Duplicate cultures.

^b)    Ethanol.

^c)   The test item precipitated in the culture medium.

 

Chromosome Aberrations in Human Lymphocyte Cultures Treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in the Absence of S9-Mix in the Second Cytogenetic Assay (24 H Exposure Time, 24 H Fixation Time)

Conc	Ethanol (0.5% v/v)				10 µg/mL				200 µg/mL				400 µg/mL				MMC-C 0.2 µg/mL
Culture	A    B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				91				79				67				44
No. of Cells scored	150  150300				150  150300				150  150300				150  150300				150  150300
No. of Cells with aberrations (+ gaps) a)	0	0		0	0	1		1	1	0		1	0	0		0	34	30		****^) 64
No. of Cells with aberrations (- gaps)	0	0		0	0	1		1	0	0		0	0	0		0	34	30		****^) 64
g’									1									1
g”
b’						1											18	9
b”																	4	6
m’
m”
exch.																	24	19
dic
d’
misc.
total aberr (+ gaps)	0	0			0	1			1	0			0	0			46	35
total aberr (- gaps)	0	0			0	1			0	0			0	0			46	34

^a)    Abbreviations used for various types of aberrations are listed inAppendix 2.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)    Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001, **** P < 0.0001.

 

Historical Control Data for in vitro Chromosome Aberration Studies of the Solvent Control

	3 hours exposure				24 hours exposure
	Gaps included		Gaps excluded		Gaps included	Gaps excluded
	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	-S9 mix
Number of aberrant cells (per 300 cells)	1.2	1.0	1.0	0.9	1.0	0.9
SD	1.5	1.4	1.2	1.2	1.3	1.3
n	64	64	64	64	61	61
Lower Control Limit (95% Control Limits)	-2	-2	-1	-2	-2	-2
Upper Control Limit (95% Control Limits)	4	4	3	3	4	3

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between May 2018 and May 2021.

 

Historical Control Data for in vitro Chromosome Aberration Studies of the Positive Control Substances

	3 hours exposure				24 hours exposure
	Gaps included		Gaps excluded		Gaps included	Gaps excluded
	-S9 mix	+S9 mix	-S9 mix	+S9 mix	-S9 mix	-S9 mix
Number of aberrant cells (per 300 cells)	64.9	54.0	63.6	52.4	85.5	83.9
SD	28.2	19.6	28.2	19.6	39.9	40.4
n	65	64	65	64	61	61
Lower Control Limit (95% Control Limits)	10	16	9	14	8	5
Upper Control Limit (95% Control Limits)	120	92	119	91	164	163

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between May 2018 and May 2021.

Appendix 2

Definitions of Chromosome Aberrations Scored in Metaphase Portraits

Aberration	Abbreviation	Description
Chromatid gap	g'	An achromatic lesion which appears as an unstained region in the chromatid arm, the size of which is equal to or smaller than the width of the chromatid and the apparently "broken" segments of the chromatid arm are in alignment.
Chromosome gap	g"	An achromatic lesion which appears as an unstained region in both chromatids at the same position, the size of which is equal to or smaller than the width of the chromatid and the apparently "broken" segments of the chromatids are in alignment.
Chromatid break	b'	An achromatic lesion in a chromatid arm, the size of which is larger than the width of the chromatid. The broken segments of the chromatid arm are aligned or unaligned.
Chromosome break	b"	An achromatic lesion in both chromatids at the same position, the size of which is larger than the width of the chromatid. The broken segments of the chromatids are aligned or unaligned.
Chromatid deletion	d'	Deleted material at the end of a chromatid arm
Minute	m'	A single, usually circular, part of a chromatid lacking a centromere.
Double minutes	m"	Two, usually circular, parts of a chromatid lacking a centromere.
Dicentric chromosome	dic	A chromosome containing two centromeres.
Tricentric chromosome	tric	A chromosome containing three centromeres.
Ring chromosome	r	A ring structure with a distinct lumen.
Exchange figure	exch.	An exchange(s) between two or more chromosomes resulting in the formation of a tri- or more-armed configuration.
Chromosome intrachange	intra	A chromosome intrachnage is scored after rejoining of a lesion within one chromosome.
Pulverized chromosomes	p	A fragmented or pulverized chromosome.
Multiple aberrations	ma	A metaphase spread containing ten or more of the above mentioned aberrations (chromatid and chromosome gaps not included). ma is counted as 10 aberrations.
Polyploidy	poly	A chromosome number that is a multiple of the normal diploid number.
Endoreduplication	endo	A form of polyploidy in which each centromere connects two or four pairs of chromatids instead of the normal one pair.

 

Conclusions:

In conclusion, this test is valid and Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Executive summary:

The objective of the study was to evaluate Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

 

The possible clastogenicity of the test item was tested in two independent experiments.

 

The study procedures described in this report are in compliance with the most recent OECD guideline.

 

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, for the lowest dose group and the highest dose group, were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).For the intermediate dose group the concentration in the dose formulation was just above the target concentration. (i.e. 111%).Because the result was only slightly above the specification and the samples were diluted from the highest dose group, which was within specification, the results are accepted. In the vehicle, no test item was detected. The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

 

In the first cytogenetic assay, the test item was tested up to 313 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix. The test item precipitated in the culture medium at this dose level after 3 h exposure.

 

In the second cytogenetic assay, the test item was tested up to 400 µg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9-mix. The test item precipitated in the culture medium at this dose level after 24 h exposure.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 

No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. 

 

In conclusion, this test is valid and Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not clastogenic in human lymphocytes under the experimental conditions described in this report. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Apr 2021 to 03 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guideline 490. “Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene", (adopted 29 July 2016).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.17: "Mutagenicity – In Vitro Mammalian Cell Gene Mutation Test”. Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Test System L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale Recommended test system in international guidelines (e.g. OECD).
Source American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in the ultra-low freezer set to maintain -150°C. The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/mL.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and
ß-naphthoflavone (100 mg/kg body weight).

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL
S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without and with S9-mix: 1.2, 2.4, 4.9, 9.8, 20, 39, 78, 156, 313, 625 and 1250 μg/mL
The dose levels selected to measure mutant frequencies at the TK-locus were: Without and with S9-mix: 9.8, 20, 39, 78, 156, 313, 325 and 1250 μg/mL exposure medium.

Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for the second mutagenicity testing: 0.98, 2.0, 3.9, 7.8, 16, 31, 125, 250 and 500 µg/mL exposure medium.
The dose levels selected to measure mutant frequencies at the TK-locus were: 0.98, 2.0, 3.9, 7.8, 16, 31 and 63 µg/mL exposure medium.

Vehicle / solvent:

The vehicle of the test item was ethanol (Extra pure, Merck, Darmstadt, Germany). A non-GLP solubility test was performed for the method development. In this solubility test, Milli-Q water, DMSO and ethanol were tested. The test item was insoluble in Milli-Q water or DMSO and dissolved in ethanol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Cleansing
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in growth medium containing 10-4 M hypoxanthine (Sigma), 2 x 10e-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10e-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on growth medium containing hypoxanthine and thymidine only. After this period cells were returned to growth medium for at least 1 day before starting the experiment.

Dose-range Finding Test
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10e6 cells (10e6 cells/mL for 3 hour treatment) or 6 x 10e6 cells
(1.25 x 10e5 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix.
The highest tested concentration was 2500 µg/mL exposure medium.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium.
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium. The cells in the final suspension were counted with the coulter particle counter.
The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hour treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10e5 cells/mL were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

Mutagenicity Test
Eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. Except in the second experiment in which seven dose levels were tested.
Since the test item was not toxic and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations may show a slight to heavy precipitate in the exposure medium.
Initially, a mutation experiment was performed with a 3 hour treatment period in the absence and presence of S9-mix up to the dose level of 2500 μg/mL. Since not enough dose levels could be selected below the highest precipitating concentration for the determination for the mutagenicity frequency (according to the OECD 490 guideline), this experiment was rejected, and no data is reported of this initial experiment. A repeat experiment for the 3 hour treatment period was performed.

Treatment of the Cells
Per culture 8 x 10e6 cells (10e6 cells/mL for 3 hour treatment) or 6 x 10e6 cells (1.25 x 10e5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test item in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium.
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium. The cells in the final suspension were counted with the coulter particle counter.

Expression Period
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10e6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutant frequency (MF).

Determination of the Mutant Frequency
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutant frequency (MF) a total number of 9.6 x 10e5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Analysis of Results
Determination of the Mutant Colonies
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutant frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces an MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutant frequency of MF(controls) + 126.

Statistics:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutant frequency in the solvent control is ≥ 50 per 10e+6 survivors and ≤ 170 per 10e+6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutant frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10e-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10e-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10e-6).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 156 to 2500 µg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 2500 μg/mL compared to the solvent control.
No toxicity in the relative suspension growth was observed up to test item concentrations of 2500 μg/mL compared to the solvent control.

First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test: Without and with S9-mix: 1.2, 2.4, 4.9, 9.8, 20, 39, 78, 156, 313, 625 and 1250 μg/mL exposure medium.

Evaluation of toxicity
The dose levels selected to measure mutant frequencies at the TK-locus were: Without and with S9-mix: 9.8, 20, 39, 78, 156, 313, 325 and 1250 μg/mL exposure medium.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 117% compared to the total growth of the solvent controls.
In the presence of S9-mix, the relative total growth of the highest test item concentration was 149% compared to the total growth of the solvent controls.

Evaluation of the mutagenicity
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second Mutagenicity Test
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.98, 2.0, 3.9, 7.8, 16, 31, 125, 250 and 500 µg/mL exposure medium.

Evaluation of toxicity
The dose levels selected to measure mutant frequencies at the TK-locus were: 0.98, 2.0, 3.9, 7.8, 16, 31 and 63 µg/mL exposure medium.
The relative total growth of the highest test item was 77% compared to the total growth of the solvent controls.

Evaluation of mutagenicity
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls

Dose-range Finding Test: Cytotoxicity of the test item (3 Hour Treatment)

Dose (µg/mL)	Cell count after 24 hours of subculture (10^5cells/mL)	Cell count after 48 hours of subculture (10^5cells/mL)	SG	RSG (%)
Without metabolic activation
SC 156 313 625 1250^(1) 2500^(1)	5.8 6.2 6.3 6.3 6.1 5.4	7.3 7.3 7.3 7.1 7.2 7.4	21 23 23 22 22 20	100 107 109 106 104 94
With metabolic activation
SC 156 313 625 1250^(1) 2500^(1)	5.3 4.9 4.7 5.0 4.9 5.2	8.3 8.1 7.8 7.9 8.1 8.1	22 20 18 20 20 21	100 90 83 90 90 96

All calculations were made without rounding off.

SC       =         solvent control 

SG     =        suspension growth       

RSG   =        relative suspension growth

⁽¹⁾        =       the test item precipitated in the exposure medium

SG = Suspension growth =[Cell count after 24 hour of subculture (Day 1) /1.6 x 10⁵]x [Cell count after 48 hours of subculture (Day 2)/1.25 x 10⁵]

RSG= [SG_(test)/SG_(control)] x 100

 

Dose-range Finding Test: Cytotoxicity of the test item (24 Hour Treatment)

Dose (µg/mL)	Cell count after 24 hours of subculture (10^5cells/mL)	Cell count after 48 hours of subculture (10^5cells/mL)	SG	RSG (%)
Without metabolic activation
SC 156^(1) 313^(1) 625^(1) 1250^(1) 2500^(1)	12.4 11.5 10.2 10.7 11.9 12.9	6.3 5.0 7.0 6.9 6.3 6.5	50 37 46 47 48 54	100 74 91 95 96 107

All calculations were made without rounding off.

SC       =         solvent control 

SG     =        suspension growth       

RSG   =        relative suspension growth

⁽¹⁾        =       the test item precipitated in the exposure medium

SG = Suspension growth = [Day 0 cell count/1.25 x 10⁵] x [Day 1 cell count/1.25 x 10⁵]

RSG= [SG_(test)/SG_(control)] x 100

 

Experiment 1: Cytotoxic and Mutagenic Response of the test item in the Mouse Lymphoma L5178Y Test System

Dose (µg/mL)	RSG (%)	CEday2 (%)	RCE (%)	RTG (%)	Mutant frequency per 10^6survivors
					Total	(	Small	Large	)
Without metabolic activation 3 hour treatment
SC SC 9.8 20 39 78 156 313 625 1250^(1) MMS	100 113 106 119 101 103 97 105 100 75	99 89 93 108 85 98 105 110 98 110 64	100 98 115 90 104 112 117 104 117 68	100 111 122 107 105 116 114 110 117 51	93 132 89 112 124 101 100 86 90 102 1225	( ( ( ( ( ( ( ( ( ( (	30 61 40 57 42 37 40 37 31 42 673	59 63 46 48 76 58 54 46 56 54 355	) ) ) ) ) ) ) ) ) ) )
With metabolic activation 3 hour treatment
SC SC 9.8 20 39 78 156 313 625 1250^(1) CP	100 138 134 130 134 119 115 103 139 81	89 89 93 101 107 105 90 107 89 95 54	100 104 114 120 119 101 120 100 107 61	100 144 152 156 159 120 139 103 149 49	123 154 155 128 20 112 103 108 156 100 1657	( ( ( ( ( ( ( ( ( ( (	43 39 58 45 31 51 48 37 52 37 651	74 107 86 74 45 54 50 66 94 58 648	) ) ) ) ) ) ) ) ) ) )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent Control = Ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

⁽¹⁾= the test item precipitated in the exposure medium

 

Experiment 2: Cytotoxic and Mutagenic Response of the test item in the Mouse Lymphoma L5178Y Test System

Dose (µg/mL)	RSG (%)	CEday2 (%)	RCE (%)	RTG (%)	Mutant frequency per 10^6survivors
					Total	(	Small	Large	)
Without metabolic activation 24 hour treatment
SC SC 0.98 2.0 3.9 7.8 16 31 63^(1) MMS	100 98 97 98 93 100 92 82 92	116 102 104 95 91 90 105 104 102 86	100 95 87 84 82 96 95 94 79	100 93 85 82 77 96 88 77 73	67 159 111 125 167 155 112 109 119 1017	( ( ( ( ( ( ( ( ( (	24 82 49 64 63 60 65 43 71 341	40 64 56 54 92 85 41 60 40 463	) ) ) ) ) ) ) ) ) )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent Control = Ethanol; MMS = Methylmethanesulfonate

⁽¹⁾= the test item precipitated in the exposure medium

 

 

Historical Control Data of the Spontaneous Mutant Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutant frequency per 10^6survivors
	-S9 Mix		+S9 mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	100	99	100
SD	28	28	28
n	96	87	93
Lower Control Limit (95% Control Limits)	45	44	46
Upper Control Limit (95% Control Limits)	154	153	154

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between May 2018 and May 2021.

 

Historical Control Data of the Mutant Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutant frequency per 10^6survivors
	-S9 Mix		+S9 mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	961	776	1154
SD	408	236	648
n	94	89	93
Lower Control Limit (95% Control Limits)	162	313	-116
Upper Control Limit (95% Control Limits)	1761	1239	2425

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between May 2018 and May 2021

Conclusions:

In conclusion, Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of the study was to evaluate the mutagenic potential of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

 

The test was performed in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. 

 

The study procedures described in this report were based on the most recent OECD guideline.

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

 

In the first experiment, the test item was tested at concentrations up to 1250 µg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. No toxicity was observed at these dose levels in the absence and presence of S9-mix. The test item precipitated in the culture medium at the highest dose level of 1250 µg/mL.

 

In the second experiment, the test item was tested at concentrations up to 63 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at these dose levels in the absence and presence of S9-mix. The test item precipitated in the culture medium at the highest dose level.

 

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

 

For the dose formulation samples at the high concentration level, the mean accuracy was below the target concentration (i.e. 89% of target). Since at this highest concentration precipitation was observed, the maximum required dose level according to the OECD 490 guideline is met and this study plan deviation did not impact the study results.

 

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

 

Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

 

The mutant frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay.

 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

 

In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency.

 

In conclusion, Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity - in vitro (Ames Assay) 

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

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. 

 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. 

Genetic toxicity - in vitro (chromosome aberration) 

The possible clastogenicity of the test item was tested in two independent experiments.

The concentrations analyzed in the dose formulation samples, for the lowest dose group and the highest dose group, were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).For the intermediate dose group the concentration in the dose formulation was just above the target concentration. (i.e. 111%).Because the result was only slightly above the specification and the samples were diluted from the highest dose group, which was within specification, the results are accepted. In the vehicle, no test item was detected. The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

 In the first cytogenetic assay, the test item was tested up to 313 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix. The test item precipitated in the culture medium at this dose level after 3 h exposure.

 In the second cytogenetic assay, the test item was tested up to 400 µg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9-mix. The test item precipitated in the culture medium at this dose level after 24 h exposure.

 The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. 

 In conclusion, this test is valid and Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not clastogenic in human lymphocytes under the experimental conditions described in this report. 

Genetic toxicity - in vitro (mouse lymphoma assay) 

The objective of the study was to evaluate the mutagenic potential of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. 

In the first experiment, the test item was tested at concentrations up to 1250 µg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. No toxicity was observed at these dose levels in the absence and presence of S9-mix. The test item precipitated in the culture medium at the highest dose level of 1250 µg/mL.

In the second experiment, the test item was tested at concentrations up to 63 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at these dose levels in the absence and presence of S9-mix. The test item precipitated in the culture medium at the highest dose level.

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

For the dose formulation samples at the high concentration level, the mean accuracy was below the target concentration (i.e. 89% of target). Since at this highest concentration precipitation was observed, the maximum required dose level according to the OECD 490 guideline is met and this study plan deviation did not impact the study results.

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

Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

The mutant frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency.

In conclusion, Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the above considerations, the substance does not need to be classified according to the CLP Regulation (EC) 1272/2008.