Fatty acids, coco, esters with 1,3-butanediol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (key, OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2

Mammalian cell micronucleus test (key, OECD 487): negative in primary human lymphocytes with and without metabolic activation.

Gene mutation in mammalian cells (key, OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

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:

01 Sep 2004 - 13 Oct 2004

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:

adopted in 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2000

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon for Salmonella strains
trp operson for E. coli strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix (10% S9 fraction) provided by MOLTOX TM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 -USA) - no further information in study report given

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500, and 5000 µg/plate.
No cytotoxicity seen up to top dose of 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: paraffin oil;
- Justification for choice of solvent/vehicle: not stated

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine, ß-Propiolactone, cis-Platinum (II) Diammine Dichloride, Dimethyl- benzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); Experiment 2 was done with pre-incubation
For each of the strains, 100 µL of the bacterial suspension (1-5 x 10e9 bacteria/mL) and 100 µL of the test substance were mixed with 2.0 mL of overlay agar and poured over the surface of a minimal agar plate (90 mm in diameter) (n = 3). The overlay agar was allowed to solidify before incubation.

DURATION
- Preincubation period: Experiment 1 was done without pre-incubation, Experiment 2 was done with pre-incubation (test substance preincubated with the test strain, and 500 µL of S9-mix fraction for 1 hour at 37° C prior to mixing with the overlay agar)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates in each of the 2 experiments
NUMBER OF ASSAYS: 2 per bacteria strain

DETERMINATION OF CYTOTOXICITY
Reduction in the number of spontaneous revertants.

Bacteriostatic activity was tested in Salmonella strain TA100 in two different assays with three replicates each. 100 µL of the bacterial suspension (1-5x10e9 bacteria/mL) and the different concentrations of the test substance were added to 2 mL of overlay agar at 45°C, containing 10% (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogeneization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48 hours at 37° C, and the number of colonies counted. A negative control containing the solvent alone was run in parallel.

For test substances that were cytotoxic already below 5000 µg/plates, the highest concentration to be retained was that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring.

Evaluation criteria:

The result of the test is considered positive if a concentration - related increase is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into account for a given concentration of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli WP2(uvrA) (pKM 101), and three fold for TA 1535 and TA 1537.

Statistics:

Descriptive statistics (mean, standard error, R)

Key result

Species / strain:

S. typhimurium, other: TA98, 100, 1535, and 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not stated
- Precipitation: not stated

HISTORICAL CONTROL DATA
- Positive historical control data (means and standard deviation): The historical values of the laboratory (January 2003 - December 2003) were within the expected range for all five strains tested with and without S9 (with and without pre-incubation with S9).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Salmonella strain TA 100 was successfully tested for absence of any bacteriostatic activity at doses ranging from 50 to 5000 µg/plate in two assays

Table 2: Mean (triplicates) number of revertant colonies ±standard error

Experiment I	without S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle	12±1.0	2.67±2.08	15.67±1.53	83.67±8.08	64.33±8.33
Negative control	11±2.0	3.67±1.15	14.33±1.53	80.67±3.79	62.33±7.02
5000	7.67±2.08	3.33±0.58	11.33±3.21	49±18.33	30±8
1500	11.0±1.0	3.33±0.58	16.33±3.51	46.33±10.26	55.33±14.29
500	8.67±0.58	3.33±1.15	16.33±2.52	70.33±13.2	51.67±10.02
150	11.33±1.15	3.67±1.53	18.0±2.0	113±14.73	59.33±24.83
50	14.0±4.58	2.67±0.58	14.67±1.15	105.33±5.51	61.33±21.78
beta propiolactone [50 µg in 10 µL]	76.33±12.06
9-aminoacridine [50 µg in 20 µL]		536.67±106.93
2-nitrofluorene [2 µg in 20 µL]			807.67±164.63
Sodium azide [20 µg in 20 µL]				915.33±135.66
cis-platinum diamine dichloride [1 µg in 10 µL]					636.33±61.45

 

Experiment I	with S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle	12.67±2.08	3.0±1.73	22.0±2.0	96.33±13.8	126±10.82
Negative control	13.33±2.08	4.0±1.0	22.0±2.0	105.33±8.74	145±15.72
5000	11.0±2.0	3.0±1.0	12.33±3.06	71±11.53	46±9.54
1500	16.33±4.62	2.33±0.58	19.0±2.0	93.33±7.77	64.33±17.9
500	9.33±0.58	3.67±1.15	24.67±19.4	80.33±6.03	106±32.05
150	12.67±2.08	4.0±1.0	25.67±2.08	115.67±7.57	124±50.21
50	13.0±1.0	5.0±2.0	29.33±4.16	130.67±12.06	123.67±42.44
2-anthramine [2 µg in 20 µL]	72.0±11.14	51.33±8.62	908.33±108.64	821.67±30.73
Dimethylbenzanthracene [5 µg in 5 µL]					699.33±108.82

 

Experiment II	without S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle (ethanol)	12.67±1.53	2.33±0.58	17±2	93±3	72.67±8.74
Negative control	14.67±2.08	3.67±0.58	15.33±2.52	85±6.56	70.33±12.5
5000	8.33±3.06	4.0±5.2	13.67±0.58	96.67±14.57	65.67±13.32
1500	8.67±2.52	4.0±5.2	14.67±2.08	83.33±4.04	68.33±18.77
500	11.33±3.21	3.0±1.0	14.67±2.0	96.67±8.33	99.67±11.93
150	11.67±1.53	2.33±0.58	15.0±2.0	104.67±13.5	72.33±16.86
50	11.0±2.65	2.0±1.0	14.33±1.53	95.67±9.45	71.0±14.11
beta propiolactone [50 µg in 10 µL]	70.67±16.2
9-aminoacridine [50 µg in 20 µL]		667.67±169.52
2-nitrofluorene [2 µg in 20 µL]			826.67±63.81
Sodium azide [20 µg in 20 µL]				768±118.19
cis-platinum diamine dichloride [1 µg in 10 µL]					523.33±148.44

 

Experiment II	with S9
	TA1535	TA1537	TA98	TA100	WP2 uvrA pKM101
Vehicle (ethanol)	11.67±3.06	2.67±1.15	23±1	118.33±3.06	128±10.82
Negative control	14.0±3.0	4.0±1.0	20±2	117±7.21	137±6.24
5000	7.33±4.04	4.0±5.2	15±2	102±9.85	95±7.21
1500	10.0±2.65	4.33±4.93	18.33±3.51	128.33±13.58	149.67±13.01
500	11.0±3.0	1.67±0.58	20.67±4.04	107.67±28.73	135±50.12
150	8.0±1.0	1.67±0.58	24.67±3.21	114.33±14.01	157.67±14.29
50	12.3±1.53	3.0±1.0	22.67±2.52	101.33±7.77	147.33±6.66
2-anthramine [2 µg in 20 µL]	63.67±5.51
2-anthramine [1 µg in 20 µL]		53.33±4.04
2-anthramine [1 µg in 10 µL]			481.33±81.68	823.33±94.71
Dimethylbenzanthracene [2.5 µg in 5 µL]					599.33±76.06

 

Conclusions:

No mutagenic potential in any of the 5 bacterial strains tested seen in 2 independent experiments with 3 replicates each.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun 2017 - 28 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministere de l'economie, Cedex, France

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: young, healthy, non-smoking donors
- Cell cycle length: 12-14 hours
- Sex, age and number of blood donors if applicable: 18 to 35 years old, 1 donor per experiment
- Whether whole blood or separated lymphocytes were used if applicable: To prepare each culture, 0.4 mL of heparinized human whole blood was added to 8 mL of culture medium containing phytohemagglutinin. The cultures were then placed at 37°C for 44 to 48 hours.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was RPMI 1640 medium (HEPES-buffered) containing 20% fetal calf serum, L glutamine (2 mM), antibiotic and antimycotic.
- Properly maintained: yes

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

0, 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL in the first experiment with and without S9 mix,
0, 156.3, 312.5, 625, 937.5 and 1250 µg/mL in the second experiment with and without S9 mix.
In both experiments, an emulsion was observed in the culture medium at the dose level of 1250 µg/mL at the end of the 3-hour treatment period, whereas no emulsion was observed in the culture medium at the end of the 24-hour treatment period, at any dose levels.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: solubility.
The test item was diluted in the vehicle at the concentration of 500 mg/mL both for the preliminary cytotoxicity test and for the cytogenetic experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine at 0.1 µg/mL without S9 for 3 hour-exposure

Details on test system and experimental conditions:

A preliminary cytotoxicity test was performed in presence and absence of S9 mix, with six dose levels of the test item and the vehicle control.
After a preliminary cytotoxicity test, the test item, diluted in ethanol, was tested in two independent experiments.

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 3h, 24h (see Table 1 below)
- Fixation time (start of exposure up to fixation or harvest of cells): 3h treatment: 27h, 24 h treatment: 48 h

CYTOKINESIS INHIBITOR: For the 24-hour treatments or for the recovery period following the 3-hour treatments, Cytochalasine B (CytoB) dissolved in dimethylsulfoxide was added in each culture to reach a final concentration of 6 µg/mL. This treatment leads to the formation of binucleated cells, by preventing separation of daughter cells after mitosis. The micronucleus analysis was then only performed in binucleated cells.

STAIN (for cytogenetic assays): Giemsa.

NUMBER OF REPLICATIONS: 2 independent experiments with 2 cultures per experiment

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At harvest, the cells were submitted to a hypotonic treatment to induce cells swelling, fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides

NUMBER OF CELLS EVALUATED: 2000 Binucleated Cells per concentration
The micronucleus analysis was performed "blind", under a microscope. Micronuclei (MN) were analyzed in 1000 Binucleated Cells (BC) per culture.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The Binucleated Cells (BC) selected for micronucleus analysis must meet the following criteria:
- cells should have two nuclei situated within the same cytoplasmic boundary,
- the two nuclei of BC should be approximately equal in size and staining, BC should have intact and distinguishable nuclear and cytoplasmic membranes.

Among the Binucleated Cells, Micronucleated Binucleated Cells (MNBC) were scored according to the following criteria:
- micronuclei should be within the same cytoplasmic boundary as the two main nuclei and clearly surrounded by a nuclear membrane,
- micronuclei should be round or oval in shape,
- the micronucleus diameter should be less than one-third of the diameter of the main nuclei (i.e. the micronucleus area should be less than 1/9th of the area of one of the main nuclei),
- micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
- micronuclei should have similar staining intensity to that of the main nuclei (or occasionally more intense),
- micronuclei should not be linked to the main nuclei via nucleoplasmic bridges,
- micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
-only BC with a number of micronuclei ≤ 5 were scored to exclude apoptosis and nuclear fragmentation.

DETERMINATION OF CYTOTOXICITY
The Replication Index (RI) was evaluated in each culture and cytotoxicity was assessed by the observation of decrease in the RI relative to vehicle control cultures. Three appropriate test item dose levels for the scoring of micronuclei were selected mainly on the basis of the cytotoxicity (i.e. achieved reduction of the RI) and on the presence of precipitate/emulsion.

Evaluation criteria:

Acceptance criteria
Each main experiment was considered valid if the following criteria were met:
- the mean frequency of cells that have undergone mitosis (binucleated + multinucleated cells) in the vehicle control cultures is at least 50%,
- the mean background frequency of Micronucleated Binucleated Cells in the vehicle control cultures should be consistent with the historical vehicle control range for the Laboratory,
- a statistically significant increase in the frequency of MNBC has to be obtained in the positive controls over the background frequency of the vehicle control cultures.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential if, in any of the experimental conditions examined, all the following criteria are met:
- a statistically significant increase in the frequency of MNBC, in comparison to the corresponding vehicle control, is obtained at one or more dose levels,
- a dose-response relationship (dose-related increase in the frequency of MNBC) is demonstrated by a statistically significant trend test,
- for at least one dose level, the frequency of MNBC of each replicate culture is above the corresponding vehicle historical range.
Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response are met.
- When the criteria of a positive response were only partially met, results were evaluated on a case by case basis, taking into account other parameters such as reproducibility between experiments.

Statistics:

Treated cell cultures were compared to that of the vehicle control cell cultures. Unless treated culture data were lower than or equal to the vehicle control data, the statistical comparison was performed using the x2 test, in which p = 0.05 was used as the lowest level of significance.
To assess the dose-response trend, a linear regression was performed between the mean frequencies of Micronucleated Binucleated Cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

lymphocytes: human

Remarks:

primary cell culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

as indicated by decrease in the replication index

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At the highest tested dose level, i.e. 1250 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 305 mOsm/kg water (327 mOsm/kg water for the vehicle control). Therefore, none of the selected dose levels was considered to produce extreme culture conditions and the dose level of 1250 µg/mL could be selected as the highest dose level for the main experiments.

Based on available solubility data, the highest achievable dose level to be used in the preliminary cytotoxicity test was 1250 µg/mL, dose level limited by the solubility of the test item in the vehicle and by the maximum treatment volume (i.e. 0.25% (v/v)). Thus, using a test item concentration of 500 mg/mL in the vehicle (i.e. ethanol) and the maximum treatment volume in the culture medium, the dose levels selected for the treatment of the preliminary test were: 2.5, 25, 125, 250, 625 and 1250 µg/mL.

At the end of both 3- and 24-hour treatment periods, an emulsion was observed in the culture medium at the dose level of 1250 µg/mL.

CYTOKINESIS BLOCK (if used) : Cytochalasin B
- Distribution of mono-, bi- and multi-nucleated cells: Given by replication index (RI)
RI=Number of binucleated cells +2(number of multinucleated cells) / total number of cells

NUMBER OF CELLS WITH MICRONUCLEI
The mean frequency of cells that have undergone mitosis (binucleated cells (BC) + multinucleated (MN) cells), as well as the mean background frequency of MNBC for the vehicle control were as specified in the acceptance criteria. Also, positive control cultures showed statistically significant increases in the frequency of MNBC. The study was therefore considered to be valid.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes, see attachment
- Negative (solvent/vehicle) historical control data: yes, see attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Following the 3-hour treatment without S9 mix, a slight to moderate cytotoxicity was observed at dose levels ≥ 625 µg/mL, as shown by a 32 to 59% decrease in the RI.
Following the 24-hour treatment without S9 mix, no cytotoxicity was induced at any dose levels, as shown by the absence of any noteworthy decrease in the RI, in either experiment.
In the presence of S9 mix, a moderate cytotoxicity was observed at the dose level of 1250 µg/mL in the first experiment, as shown by a 51% decrease in the RI, whereas no cytotoxicity was observed at any dose levels in the second experiment, as shown by the absence of any noteworthy decrease in the RI.

Following the 24-hour treatment without S9 mix and the 3-hour treatment with S9 mix,statistically significant increases in the frequency of MNBC were noted in the first experiment at the highest tested dose level of 1250 µg/mL relative to the corresponding vehicle control. However, no dose-response relationship was demonstrated by the linear regression in either test condition. Since the criteria for a positive response were partially met in both test conditions, a second experiment was performed under the same experimental conditions.

In the second experiment,neither statistically significant nor dose-related increase in the frequency of MNBC was noted at any of the analyzed dose levels relative to the corresponding vehicle control. Moreover,none of the analyzed dose levels showed frequency of MNBC of both replicate cultures above the corresponding vehicle reference data range,in either test condition.

Since the increases observed in the first experiment were not reproduced in this second experiment despite the narrower range of dose levels used, they were considered as non-biologically relevant.

Table 2: Experiment I: 3 hours exposure without S9 mix + 24-hour recovery prior preparation

		RI in culture 1, culture 2	Mean Decrease in RI in %	Micro-nucleated cells in %
Vehicle control (ethanol)		0.92, 0.91		2.0
Test item 39.06 µg/mL		0.83, 0.85	8	not evaluated
Test item 78.13 µg/mL		0.86, 0.86	6	not evaluated
Test item 156.3 µg/mL		0.81, 0.84	10	not evaluated
Test item 312.5 µg/mL		0.73, 0.75	19	3.0
Test item 625 µg/mL		0.58, 0.67	32	2.5
Test item 1250 µg/mL	E	0.23, 0.53	59	3.5
Colchicine 0.1 µg/mL		0.49, 0.55	43	9.5**

Table 3: Experiment I: 3 hours exposure with S9 mix + 24-hour recovery prior preparation

		RI in culture 1, culture 2	Mean Decrease in RI in %	Micro-nucleated  cells in %
Vehicle control (ethanol)		0.88, 0.92		0.0
Test item 39.06 µg/mL		0.90, 0.88	1	not evaluated
Test item 78.13 µg/mL		0.97, 086	none	not evaluated
Test item 156.3 µg/mL		0.90, 0.86	2	not evaluated
Test item 312.5 µg/mL		0.77, 0.85	10	1.0
Test item 625 µg/mL		0.75, 0.75	17	0.5
Test item 1250 µg/mL	E	0.46, 0.43	51	4.5**
Cyclophoshamide 6 µg/mL		0.61, 0.57	34	10.5***

Table 4: Experiment II: 3 hours exposure with S9 mix + 24-hour recovery prior preparation

		RI in culture 1, culture 2	Mean Decrease in RI in %	Micro-nucleated cells in %
Vehicle control (ethanol)		0.80, 0.78		3.5
Test item 156.3 µg/mL		0.78, 0.83	none	not evaluated
Test item 312.5 µg/mL		0.82, 0.84	none	not evaluated
Test item 625 µg/mL		0.80, 0.85	none	4.0
Test item 937.5 µg/mL		0.79, 0.73	4	1.5
Test item 1250 µg/mL	E	0.77, 0.63	11	1.0
Cyclophoshamide 6 µg/mL		0.55, 0.58	28	9.5*

Table 5: Experiment I: 24 hours exposure without S9 mix

		RI in culture 1, culture 2	Mean Decrease in RI in %	Micro-nucleated cells in %
Vehicle control (ethanol)		0.90, 0.88		1.5
Test item 39.06 µg/mL		0.86, 0.86	3	not evaluated
Test item 78.13 µg/mL		0.80, 0.79	11	not evaluated
Test item 156.3 µg/mL		0.80, 0.71	15	not evaluated
Test item 312.5 µg/mL		0.74, 0.78	14	2.5
Test item 625 µg/mL		0.77, 0.72	16	2.5
Test item 1250 µg/mL		0.71, 0.74	18	11.5***
Mitomycin C 0.1 µg/mL		0.75, 0.74	16	22.5***

Table 6: Experiment II: 24 hours exposure without S9 mix

		RI in culture 1, culture 2	Mean Decrease in RI in %	Micro-nucleated cells in %
Vehicle control (ethanol)		0.74, 0.74		4.5
Test item 156.3 µg/mL		0.73, 0.63	9	not evaluated
Test item 312.5 µg/mL		0.75, 0.65	6	not evaluated
Test item 625 µg/mL		0.70, 0.74	3	2.5
Test item 937.5 µg/mL		0.73, 0.60	10	5.5
Test item 1250 µg/mL		0.77, 0.58	9	4.0
Mitomycin C 0.1 µg/mL		0.50, 0.54	30	21.0***

E= emulsion was noted in the culture medium at the end of treatment; RI = replication index;
*statistically significantly higher than corresponding control value (p<0.05)
**statistically significantly higher than corresponding control value (p<0.01)
***statistically significantly higher than corresponding control value (p<0.001)

 

Conclusions:

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using human lymphocytes, either in the presence or absence of a rat liver metabolizing system, up to the highest achievable dose level of 1250 µg/mL.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun 2017 - 28 Mar 2018

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:

adopted 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministere de l'economie, Cedex, France

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Manassas, USA
- Cell cycle length: 10-12 hours
- TK phenotypic expression time: 2 days
- Modal number of chromosomes: 40

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% v/v (3-hour treatment) or 10% v/v (24-hour treatment) in a 37°C, 5% carbon dioxide humidified incubator.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: Mutant cells (TK-/-) can form colonies in the selective medium (Trifluorothimidine medium), while non-mutant cells (TK+/-) cannot. Small, slow growing colonies indicate chromosome damage, large colonies indicate point mutations.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from a liver microsomal fraction of rats induced with Aroclor 1254; final concentration of 2% in medium.

Test concentrations with justification for top dose:

0, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL for both experiments with or without S9 mix.
An emulsion, which did not prevent any scoring, was observed in the culture medium at dose levels ≥ 62.5 µg/mL at the end of both treatment periods.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: Test item was found to be poorly soluble in culture medium.
The test item was diluted in the vehicle at concentrations of 500 mg/mL for the preliminary cytotoxicity test, and at 50 mg/mL for both mutagenicity experiments.

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:

In a preliminary cytotoxicity test, six dose levels (one culture/dose level) were tested both with and without metabolic activation.
After the preliminary toxicity test, the test item was tested in two independent experiments

METHOD OF APPLICATION: during treatment: in suspension; during selection time in 96-well plates
Cultures of 20 mL at 5.0E+05 cells/mL (3-hour treatments in 50 mL tubes) or cultures of 50 mL at 2.0E+05 cells/mL (24-hour treatment in flasks) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). For selection 2000 cells/well were seeded in four 96-well plates/culture (eight plates/dose level).

EXPOSURE DURATION :
Experiment I: 3 hours with S9, 3 hours without S9, 24 hours without S9
Experiment II: 24 hours without S9

- Expression time (cells in growth medium): 48 hours; during the mutant phenotype expression period, the cultures were maintained at the density of approximately 2 x 2.0E+05 cells/mL, whenever possible.
- Selection time (if incubation with a selection agent): 11-12 days;

SELECTION AGENT (mutation assays): medium with 4 µg trifluorothymidine

NUMBER OF REPLICATIONS: 2 experiments with duplicate cultures

EVALUATION:
The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.
size of small colonies: < 25% of the diameter of the well
size of large colonies: > 25% of the diameter of the well

DETERMINATION OF CYTOTOXICITY
- Method: Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2)

Evaluation criteria:

Acceptance criteria
- the Cloning Efficiency (CE2) of the vehicle controls at the end of the expression time should be between 0.65 and 1.2,
- the mutation frequency of the vehicle controls should fall within the normal range of 50.0E-6 to 170.0E-6,
- the suspension growth of the vehicle controls should be between 8 and 32 for the 3-hour treatment period, and between 32 and 180 for the 24-hour treatment period

The criteria for a positive control are as follows:
an increase above the vehicle control mutation frequency (IMF) of at least 300.0E-6, the increase in the small colony mutation frequency accounting for at least 40%,
or an increase in the small colony mutation frequency of at least 150.0E-6 above that seen in the concurrent vehicle control.

In addition, the upper limit of cytotoxicity observed in the positive control culture should have an Adj. RTG greater than 10%.

Evaluation criteria
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126.0E-6
- a dose-response relationship is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG ≥ 10%. The statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at levels of 62.5 µg/mL and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At the highest tested dose level, i.e. 2500 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 340 mOsm/kg water (391 mOsm/kg water for the vehicle control). Thus none of the selected dose levels was considered to produce extreme culture conditions.

PRELIMINARY CYTOTOXICITY TEST
Based on available solubility data, the highest selected dose level was 2500 µg/mL since it was expected to produce emulsion in the culture medium. This dose level was obtained using a test item concentration of 500 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium for the 3-hour treatments and 250 µL/50 mL culture medium for the 24-hour treatment). The dose levels selected for the treatment of the preliminary test were 5, 50, 250, 500, 1250 and 2500 µg/mL.
At the end of the treatment periods (3- or 24-hour treatment), an emulsion was observed in the culture medium at dose levels ≥ 50 µg/mL.
Following the 3- and 24-hour treatments without S9 mix as well as the 3-hour treatment with S9 mix, a slight to severe cytotoxicity was induced at dose levels ≥ 250 µg/mL, as shown by a 57 to 100% decrease in the Adj. RTG.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes, see attachment
- Negative (solvent/vehicle) historical control data: yes, see attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Following the 3-hour without S9 treatment, a moderate cytotoxicity was induced at the highest dose level of 250 µg/mL, as shown by a 67% decrease in the Adj. RTG.
Following both 24-hour treatments without S9, a slight to severe cytotoxicity was induced at dose levels ≥ 62.5 µg/mL, as shown by a 53 to 94% decrease in the Adj. RTG.
At treatment with S9 slight cytotoxicity was induced at the highest dose level of 250 µg/mL, as shown by a 52% decrease in the Adj. RTG.

With one exception per experiment, related to the 24-hour treatment without S9 mix, which was considered not to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

Table 1: Experiment I, 3-hours without S9 mix, mean values of both cultures

	µg/mL		Mean Adjusted RTG	CE	mutant colonies per 10.0E+6 cells	LC	SC	IMF
Vehicle control	0		100	0.67	146	79	60
Test item	1.95		97	0.67	147	66	76	1
Test item	3.91		95	0.68	157	77	74	11
Test item	7.81		107	0.77	116	49	65	none
Test item	15.6		84	0.71	148	75	68	3
Test item	31.3		91	0.72	132	60	67	none
Test item	62.5	E	86	0.78	126	55	65	none
Test item	125	E	63	0.63	155	71	77	9
Test item	250	E	33	0.90	133	57	71	none
methylmethane sulfonate	25		20	0.24	1397	420	778	1252

Table 2: Experiment I, 24-hours without S9 mix, mean values of both cultures

	µg/mL		Mean Adjusted RTG	CE	mutant colonies per 10.0E+6 cells	LC	SC	IMF
Vehicle control	0		100	0.67	159	78	78
Test item	1.95		111	0.72	136	73	60	none
Test item	3.91		94	0.79	110	55	51	none
Test item	7.81		143	0.84	136	68	63	none
Test item	15.6		73	0.78	168	73	88	9
Test item	31.3		121	0.86	120	53	66	none
Test item	62.5	E	41	0.72	168	75	87	8
Test item	125	E	42	0.75	116	49	64	none
Test item	250	E	11	0.60	119	60	56	none
methylmethane sulfonate	5		92	0.64	802	249	477	643

Table 3: Experiment II, 24-hours without S9 mix, mean values of both cultures

	µg/mL		Mean Adjusted RTG	CE	mutant colonies per 10.0E+6 cells	LC	SC	IMF
Vehicle control	0		100	0.88	137	66	63
Test item	1.95		115	1.12	117	55	56	none
Test item	3.91		92	0.85	163	81	72	26
Test item	7.81		92	0.85	132	59	68	none
Test item	15.6		116	1.04	160	73	75	23
Test item	31.3		63	0.82	161	81	70	24
Test item	62.5	E	47	1.08	146	78	58	9
Test item	125	E	30	0.91	138	67	63	1
Test item	250	E	6	0.90	182	77	94	45
methylmethane sulfonate	5		41	0.64	1495	410	522	1357

Table 4: Experiment I, 3-hours with S9 mix, mean values of both cultures

	µg/mL		Mean Adjusted RTG	CE	mutant colonies per 10.0E+6 cells	LC	SC	IMF
Vehicle control	0		100	0.98	155	72	76
Test item	1.95		126	1.11	154	79	64	none
Test item	3.91		145	0.94	140	72	61	none
Test item	7.81		125	0.98	146	68	69	none
Test item	15.6		129	0.99	151	73	68	none
Test item	31.3		142	1.18	164	72	78	10
Test item	62.5	E	105	0.80	181	78	93	27
Test item	125	E	94	1.10	168	69	85	13
Test item	250	E	48	1.21	202	78	107	48
cyclophosphamide	3		61	0.71	1029	309	416	875

RTG = relative total growth; E = Emulsion observed in the culture medium at the end of treatment;
CE = cloning efficiency, LC = large colonies, SC = small colonies, IMF = induced mutation frequency

 

A dose-response relationship in the mutation frequency was demonstrated by the linear regression (p < 0.005) in the experiment with S9. Since all the Induced Mutation Frequencies (IMF) remained substantially below the Global Evaluation Factor of 126.0E-6, this linear trend was considered as meaningless and these results were considered to be a negative response.

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable

Additional information

Gene mutation in bacteria

The mutagenic potential of Fatty acids, coco, esters with 1,3-butanediol (CAS 73138-39-3) was tested in a reverse mutation assay according to OECD Guideline 471 under GLP conditions (key study, 2004). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA pKM 101 were used in two experiments. Tester strains were incubated with test material at concentrations of 0, 50, 150, 500, 1500, and 5000 μg/plate with and without the addition of a metabolic activation system. No cytotoxicity or precipitation was reported. Vehicle and appropriate positive controls were included into the study design. Positive control materials induced significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent vehicle and negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, coco, esters with 1,3-butanediol (CAS 73138-39-3) did not induce point mutations by base-pair changes or frame-shifts in the genome of the bacterial strains tested.

 

Cytogenicity in mammalian cells

The mutagenic potential of Fatty acids, coco, esters with 1,3-butanediol (CAS 73138-39-3) was tested in a in vitro gene mutation study according to OECD Guideline 487 under GLP conditions (key study, 2018). Human primary lymphocytes were obtained from two healthy non-smoking donors (18-35 years old) for Experiment I and for Experiment II. Treatments started after a 44-48 hour stimulation period with phytohaemagglutinine (PHA) when cells were actively proliferating. In the first experiment, cells were exposed to the test substance for 3 hours (with and without metabolic activation), followed by 24 hours of recovery and for 24 hours continuous treatment without S9 mix (no recovery). In the second experiment cells were exposed to the test substance for 3 h with S9 mix, followed by 24 hours of recovery and for 24 hours continuous treatment without S9 mix (no recovery). For the 24-hour treatments or for the recovery period following the 3-hour treatments, Cytochalasine B (CytoB) dissolved in dimethylsulfoxide was added in each culture to reach a final concentration of 6 µg/mL. Cell harvest was after recovery or after the 24-hour continuous exposure.

Test concentrations were up to 1250 µg/mL in the both experiment with and without metabolic activation. The number of micro-nucleated cells was determined in a sample of 2000 bi-nucleated cells for each of the three highest test substance concentrations. Vehicle (solvent) controls induced frequencies within the range expected for normal human lymphocytes. Cyclophosphamide, mitomycin C and colchicine were used as positive controls and confirmed satisfactory validity of the test system.

Following the 3-hour treatment without S9 mix no statistically significant increase in the frequency of micro-nucleated binuclear cells (MNBC) was noted. Following the 24-hour treatment without S9 mix and the 3-hour treatment with S9 mix, statistically significant increases in the frequency of MNBC were noted in the first experiment at the highest tested dose level of 1250 µg/mL relative to the corresponding vehicle control. However, no dose-response relationship was demonstrated by the linear regression in either test condition. Since the criteria for a positive response were partially met in both test conditions, a second experiment was performed under the same experimental conditions in order to check the reproducibility of these increases. In the second experiment, neither statistically significant nor dose-related increase in the frequency of MNBC was noted at any of the analysed dose levels relative to the corresponding vehicle control. Moreover, none of the analysed dose levels showed frequency of MNBC of both replicate cultures above the corresponding vehicle reference data range, in either test condition.

Overall the results were considered to meet the criteria for a negative response. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

 

Genetic mutation in mammalian cells

The mutagenic potential of Fatty acids, coco, esters with 1,3-butanediol (CAS 73138-39-3) was tested in a in vitro gene mutation study according to OECD Guideline 490 under GLP conditions (key study, 2018). Duplicate cultures of mouse lymphoma L5178Y cells were evaluated. In the first experiment, the cells were treated for 3 h with 0, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, and 250 µg/mL in the presence or absence of metabolic activation (Aroclor 1254 induced rat liver S9 mix) and for 24 hours without metabolic activation. In the second experiment, same concentrations were applied without metabolic activation for 24 hours. Emulsion (precipitation) occurred at 62.5 μg/mL and above in the presence and absence of metabolic activation following 3 and 24 hours treatment. Following the 3-hour without S9 treatment, a moderate cytotoxicity was induced at the highest dose level of 250 µg/mL, as shown by a 67% decrease in the adjusted relative total growth (Adj. RTG). Following both 24-hour treatments without S9, a slight to severe cytotoxicity was induced at dose levels ≥ 62.5 µg/mL, as shown by a 53 to 94% decrease in the Adj. RTG. At treatment with S9 slight cytotoxicity was induced at the highest dose level of 250 µg/mL, as shown by a 52% decrease in the Adj. RTG. Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. Positive and negative controls were valid and in range of historical control data. No significant increase in the mutation frequency at the thymidine kinase locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

Overall conclusion for genetic toxicity

The results of the available in vitro mutagenicity and in vitro cytogenicity studies were consistently negative. Based on the available data, no hazard regarding genotoxicity is identified for Fatty acids, coco, esters with 1,3-butanediol (CAS 73138-39-3).

Justification for classification or non-classification

The available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.