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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (read-across OECD 471): negative in S. thyphimurium strains TA97a, TA98, TA100, TA102 and TA1535 with and without metabolic activation

Cytogenicity in mammalian cells (read-across OECD 473): negative in human lymphocytes and chinese hamster lung cells with and without metabolic activation

Gene mutation in mammalian cells (read-across OECD 476): negative in Chinse hamster lung fibroblasts (V79) and 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:

weight of evidence

Study period:

17 Jan - 17 Aug 2017

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: 21 Jul 1997

Deviations:

no

GLP compliance:

yes

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

other: TA1535, TA102, TA100, TA98 and TA97a

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally

Test concentrations with justification for top dose:

Dose-range-finding experiments
Experiment 1c:
50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Experiment 1d:
0.05, 0.15, 0.5, 1.5, 5 and 15 µg/plate with and without metabolic activation

Main experiments
Experiment 1e:
0.05, 0.15, 0.5, 1.5, 5 and 15 µg/plate with and without metabolic activation
Experiment 2b:
0.23, 0.47, 0.94, 1.88, 3.75, 7.5 and 15 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of the solubility of the test substance in the vehicle and the relative nontoxicity of the vehicle to the bacteria evaluated in a pre-experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

demineralised water, DMSO and ethanol (including solvents for positive controls)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: see below

Details on test system and experimental conditions:

Experiments 1a, 1b and 2a were declared invalid and were therefore not reported here.

METHOD OF APPLICATION: Experiment 1e: in agar (plate incorporation); Experiment 2b: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications per experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants and reduction in bacterial background lawn

Evaluation criteria:

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

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1e and 2b: at 15 µg/plate in all strains +/- S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed up to 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: In the dose range finding studies cytotoxicity was observed in all strains from 15 µg/plate.

Table 1. Mean revertants Experiment 1e

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	83	69	52	50	82	102	356	345	24	22
	sd	6.7	5	5.2	10.7	20.6	18.5	62.9	72.6	8	7.5
DMSO	Mean	85	78	43	39	110	101	257	324	21	21
	sd	5.6	13.6	5.5	11.9	6	21.2	26.6	20.8	0.6	5.3
Ethanol	Mean	78	70	47	39	107	127	292	258	23	22
	sd	11	5.9	3.8	10.7	6.4	3.1	58.9	24	3.6	3.1
Positive	Mean	343	657	560	487	404	1001	689	1083	265	132
Controls*	sd	35.9	24.1	146	123.3	17.4	0	60.2	400	59.5	15.2
	f(I)	4.04	8.42	13.02	12.49	4.93	9.91	2.68	3.34	11.04	6.29
15 µg/plate	Mean	14	21	2	4	22	17	165	149	1	2
	sd	6	7.2	1.2	1.7	9	4	16.8	20.2	0	1.2
	f(I)	0.18	0.3	0.04	0.1	0.21	0.13	0.57	0.58	0.04	0.09
5 µg/plate	Mean	116	68	26	37	97	86	311	387	16	21
	sd	15.6	15.7	3.5	13.7	13.5	10	49.4	31.1	4.4	1.7
	f(I)	1.49	0.97	0.55	0.95	0.91	0.68	1.07	1.5	0.7	0.95
1.5 µg/plate	Mean	84	90	32	43	104	97	309	356	25	19
	sd	2.1	18.7	8.1	11.4	26.5	8.1	32.6	69.3	4.4	2.9
	f(I)	1.08	1.29	0.68	1.1	0.97	0.76	1.06	1.38	1.09	0.86
0.5 µg/plate	Mean	82	83	30	37	94	93	360	432	18	22
	sd	12.1	25.2	9.6	6.8	9.1	14.7	34.2	52.5	2.6	1.2
	f(I)	1.05	1.19	0.64	0.95	0.88	0.73	1.23	1.67	0.78	1
0.15 µg/plate	Mean	76	86	28	40	103	92	312	259	22	22
	sd	11.9	4.4	1.5	3.2	18	10.4	60.5	78.2	3.8	3.5
	f(I)	0.97	1.23	0.6	1.03	0.96	0.72	1.07	1	0.96	1
0.05 µg/plate	Mean	79	95	27	38	103	123	337	305	25	21
	sd	25.7	21.7	6.7	10	33.8	5.8	14	98	5.6	4
	f(I)	1.01	1.36	0.57	0.97	0.96	0.97	1.15	1.18	1.09	0.95

 f(I) = increase factor

* Different positive controls were used

Table 2. Mean revertants Experiment 2b

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	83	91	33	36	88	100	289	348	17	19
	sd	8.7	5.6	5.3	5	3	15	12.2	25	6.8	3.8
DMSO	Mean	96	89	30	39	99	98	336	295	18	15
	sd	13.9	18.7	4.4	2.1	16.3	9.2	32.7	73.2	1.7	3.2
Ethanol	Mean	73	103	37	37	83	95	280	316	15	15
	sd	2.6	7	10.2	7.6	10.4	15	62.9	63.5	3.6	4.2
Positive	Mean	338	533	493	260	579	897	1235	1251	457	157
Controls*	sd	31	50.3	188.7	10.6	83.3	180.7	364.5	226.9	55.5	9.2
	f(I)	3.52	5.99	16.43	6.67	6.58	9.15	3.68	4.24	26.88	10.47
15 µg/plate	Mean	10	3	1	2	3	2	18	63	3	2
	sd	1.7	1.5	0	1	0	1	1.2	20.3	4	1.2
	f(I)	0.14	0.03	0.03	0.05	0.04	0.02	0.06	0.2	0.2	0.13
7.5 µg/plate	Mean	83	82	32	36	83	98	243	273	16	21
	sd	5.8	20.5	4.4	1.2	5.8	10.5	20.5	45.1	4.4	1.5
	f(I)	1.14	0.8	0.86	0.97	1	1.03	0.87	0.86	1.07	1.4
3.75 µg/plate	Mean	94	85	30	32	81	100	247	275	15	14
	sd	6.7	4.2	6.1	7.8	7.8	3.1	46.4	74.6	2.1	1.5
	f(I)	1.29	0.83	0.81	0.86	0.98	1.05	0.88	0.87	1	0.93
1.88 µg/plate	Mean	78	91	32	31	92	102	335	224	14	18
	sd	10.4	12.1	1.7	4.6	9.2	6.1	8.3	2	4.6	4
	f(I)	1.07	0.88	0.86	0.84	1.11	1.07	1.2	0.71	0.93	1.2
0.94 µg/plate	Mean	95	99	30	44	85	90	223	244	16	10
	sd	11	11.1	8.3	10.1	13.8	8.5	62	10.6	2.5	0.6
	f(I)	1.3	0.96	0.81	1.19	1.02	0.95	0.8	0.77	1.07	0.67
0.47 µg/plate	Mean	107	110	36	33	82	95	205	239	18	12
	sd	4.6	17.1	4	4.7	6.9	11.4	36.3	84.3	1	1.7
	f(I)	1.47	1.07	0.97	0.89	0.99	1	0.73	0.76	1.2	0.8
0.23 µg/plate	Mean	89	76	31	36	85	88	209	249	14	11
	sd	15	12.5	6	6.2	14	15.3	48.1	14	2	1
	f(I)	1.22	0.74	0.84	0.97	1.02	0.93	0.75	0.79	0.93	0.73

 f(I) = increase factor

* Different positive controls were used

Conclusions:

Under the conditions of the Ames test the substance was not mutagenic in any of the five bacterial strains (TA1535, TA98, TA100, TA102 and TA97a) tested with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 May 2010 - 12 Jun 2010

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:

adopted: 21 Jul 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

other: CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6h treatment with and without S9-mix: 0.02, 0.39, 0.078 and 0.156 mg/mL
24 and 48h treatment without S9-mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5.0 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, the test substance was insoluble in water and physiological saline. And the solubility test was performed with DMSO and acetone. The test substance was insoluble at 500.0 mg/mL in DMSO, and was dissolved at 500.0 mg/mL in acetone, and neither generation of gas nor exothermic reaction was observed. Therefore acetone was selected as solvent in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6, 24 and 48 h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid
STAIN (for cytogenetic assays): 0.1% crystal violet solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells carrying greater than 38 chromosomes including triploid were recorded as polyploidy

Evaluation criteria:

The following criteria were set to evaluate the frequency of aberrant cells in each dose group:
A final judgment was concluded excluding gaps, nevertheless, separate records were kept for both including and excluding gaps.
Negative (-) less than 5%
Equivocal (+-) 5% or more, less than 10%
Positive (+) 10% or more

Key result

Species / strain:

other: CHU/IU

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 0.078 µg/mL and higher

RANGE-FINDING/SCREENING STUDIES: In the results of the cell growth inhibition test, the dose of 50% cell growth inhibition was 5.0 mg/mL and
more all of without S9 mix, with S9 mix, 24 h and 48 h exposure. In addition, the cell growth inhibition was observed, though the cell growth rate was more than 50% at the 0.156~1.25 mg/mL doses of the 24 h exposure and the 0,313~1.25 mg/mL doses of 48 h exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Table 1. Effects of the test substance on viability and chromosome aberrations.

Test item	Concentration	Cell viability	Aberrant cells in %
	in µg/mL	in %	Numerical	Structural including gaps
Exposure period 6h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.0	1.5
MMC	0.010	105.5	0.0	39.5
Test substance	0.020	101.0	0.5	0.0
	0.039	98.0	1.0	1.5
	0.078	88.0	0.0	0.0
	0.156	82.5	0.5	1.0
Exposure period 6h with S9 mix
Untreated	--	--	0.0	2.0
Solvent	--	100	0.0	0.5
B(a)P	7.000	96.5	0.0	28.5
Test substance	0.020	96.0	0.0	2.0
	0.039	92.0	0.0	0.0
	0.078	86.0	1.0	0.5
	0.156	84.5	0.5	1.0
Exposure period 24h without S9 mix
Untreated	--	--	1.0	0.5
Solvent	--	100	0.5	0.0
MMC	0.050	102.0	0.0	41.5
Test substance	0.078	90.5	0.0	0.5
	0.156	82.5	0.0	1.0
	0.313	76.0	1.0	1.0
	0.625	66.0	0.0	0.5
	1.250	66.0	0.5	2.0
	2.500	84.5	0.5	0.0
	5.000	92.0	0.0	1.0
Exposure period 48h without S9 mix
Untreated	--	--	0.0	0.0
Solvent	--	100	0.5	0.0
MMC	0.050	94.0	0.0	58.5
Test substance	0.078	99.0	0.0	1.0
	0.156	86.5	0.0	0.5
	0.313	74.5	1.0	0.0
	0.625	70.0	0.0	0.0
	1.250	79.5	0.5	0.0
	2.500	87.5	0.5	0.0
	5.000	92.5	0.5	1.0

MMC: Mitomycin

B(a)P: Benz(a)pyrene

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Oct - 18 Apr 2002

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:

adapted: 21 Jul 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Preliminary toxicity test:
with and without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL

First experiment (and repeat tests):
without metabolic activation: 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL
without metabolic activation (repeat): 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Only slides from cultures of the following dose groups were selected for metaphase analyses:

First experiment:
without metabolic activation: 1250, 2500 and 5000 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Second experiment:
without metabolic activation (repeat): 40, 80 and 160 µg/mL
with metabolic activation: 625, 1250 and 2500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin (0.015 µg/mL, -S9), cyclophosphamide (6 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary test and first main test:
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

Second main test:
- Exposure duration: +S9: 3 h, -S9: 20 h
- Fixation time (start of exposure up to fixation of cells): 20 h (approx. 1.5 cell cycles)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 metaphases (when possible) and 1000 cells for the determination of mitotic index

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (calculated as percentage of cells in metaphases)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, defined as metaphases with multiples of the haploid chromosome number other than diploid (e.g. 3n, 4n etc.) and determined in 200 metaphases
- Determination of endoreplication: yes, defined by the presence of chromosomes with 4, 8 chromatids and determined in 200 metaphases

Evaluation criteria:

EVALUATION OF RESULTS
The study was considered as valid when:
- the negative control cultures showed a low frequency of metaphases with chromosome aberrations, normally 0 - 3% (excluding gaps)
- the positive control cultures showed a clear increase in the frequency of metaphases with chromosome aberrations

For the evaluation of the results, the number of metaphases with chromosome aberrations of each test condition were compared to the concurrent negative control. Gaps were recorded but excluded from the analyses.

The test material was considered clastogenic in this test system if all of the following criteria were met:
1. increases in the frequency of chromosome aberrations were determined at one or more test concentrations
2. reproducible increases in aberrant chromosomes between replicates
3. statistically significance in the increases of chromosome aberrations
4. increases exceed the historical negative control range
5. increases were not associated with large changes in pH or osmolarity
The evidence of a dose-response relation ship was considered to support the conclusion.

The test material was considered non-clastogenic in this test system when the increase in chromosomal aberrations was not statistically significanct and/or no reproducibility was observed.

Results which failed to meet the above mentioned criteria were considered as equivocal.

Statistics:

When appropriate, Fischer´s Exact Test was performed to evaluate statistical significance.

Key result

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related toxicity which reduced the mitotic index in the high-dose group (5000 µg/mL) to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation (table 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First main test
As the frequencies of metaphases with chromosomal aberrations were in general unacceptably high (4% for the duplicates without metabolic activation and 7 or 3% for the duplicates with metabolic activation), the repetition of the first experiment was conducted with the identical experimental design as the initial test. The values for chromosome aberrations within the test samples were between 1 and 9%, but they were not reproducible between the replicates nor did they show any dose-related effect (the data from the initial experiment are not included in the study report).
Scoring of slides prepared from the repeat of the initial experiment revealed no appropriate increases in chromosomal aberrations within the positive control samples without metabolic activation. Thus, this part of the test was considered as invalid and therefore repeated.

Second main test
As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups (data not shown), this part of the test was repeated with lower dosages in the second test (table 3).

Polyploid and endoreduplicated metaphases
Single polyploid metaphases were observed at few test points without showing a dose-relation ship. Therefore, this effect is considered as incidental and not treatment-related. In contrast, no endoreduplicated metaphases were observed.

In each test group despite the two positive controls treated with cyclophosphamide, 100 metaphases were counted. In the cyclophosphamide treated samples only 59 and 30 scorable metaphases were detected.

Test validity
The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values (table 4). The positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations in the valid parts of the tests, thereby demonstrating the sensitivity of the test and the efficacy of the S9 mix.

Table 1. Test results of the preliminary toxicity test

 

Treatment (µg/mL)	Mitotic index (MI)
	Without S9			With S9
	Individual values		Relative mean MI (%)	Individual values		Relative mean MI (%)
0	4.8	5.3	100	3.8	2.9	100
313	4.8	4.3	90	3.1	3.5	99
625	4.3	4.1	83	2.9	3.5	96
1250	3.4	3.7	70	3.4	2.8	93
2500	3.7	3.3	69	2.1	1.8	58
5000	3.6	2.9	64	0.4	0.3	10

Mitotic index: percentage of cells at metaphase

Individual values: values for each of the duplicate     

Relative mean MI: relative mean mitotic index for the duplicates

 

 

Table 2. Test results of the first main test (-S9: second repeat test, +S9: repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	1 / 0	2 / 1	1 / 0		1 / 0
1250	-	110	1 / 1	0 / 2	2 / 1
2500	-	75	1 / 4	1 / 3	1 / 4
5000	-	70	2 / 3	2 / 1	2 / 4
Daunomycin (0.015 µg/mL)	-	91	17 / 14 **	9 / 5	20 / 14	2 / 1
0	2%	100	0 / 0	1 / 2	0 / 0
625	2%	67	0 / 1	0 / 0	0 / 1
1250	2%	51	0 / 0	2 / 1	0 / 0
2500	2%	42	0 / 1	1 / 1	0 / 1
Cyclophosphamide (6 µg/mL)	2%	20	33a /19b **	16 / 3	36 / 20	15 / 9

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 3. Test results of the second main test (repeat test)

Treatment (µg/mL)	S9 mix	Relative mean MI (%)	No. aberrant metaphases	Number and types of aberrations			Number of polyploid metaphases
				Gaps	Breaks	Exchanges
0	-	100	0 / 3	1 / 5	0 / 3
40	-	84	1 / 4	3 / 6	1 / 4		1 / 0
80	-	59	1 / 2	0 / 2	1 / 2
160	-	49	0 / 2	3 / 1	0 / 2		0 / 1
Daunomycin (0.015 µg/mL)	-	102	13 / 14 **	6 / 9	13 / 13	1 / 1
0	4%	100	2 / 1	3 / 2	1 / 1	1 / 0
625	4%	66	2 / 4	3 / 1	5 / 1		0 / 1
1250	4%	61	1 / 1	3 / 4	1 / 1
2500	4%	33	5 / 1	3 / 0	3 / 4
Cyclophosphamide (6 µg/mL)	4%	77	36 / 33 **	8 / 8	37 / 33	11 / 11

Relative mean: relative mean mitotic index for the duplicates

No. of aberrant metaphases: Number of aberrant metaphases (excluding gaps) for the duplicates

** Statistically significant with p < 0.01

a: only 59 scoreable metaphases

b: only 30 scoreable metaphases

The numbers of determined metaphases, aberrations and polyploid metaphases are given for the duplicates (first sample / second sample)

 

Table 4. Historical Data (n = 9 previous study)

Treatment (µg/mL)	S9 mix	Frequency of metaphases with aberrant chromosomes excluding gaps (%)				Number of cultures
		Mean	SD	Mimimum	Maximum
Negative control	-	0.8	0.8	0	3	32
Daunomycin (0.015 µg/mL)	-	15.1	7.4	7	34	32
Negative control	+	0.7	0.9	0	3	32
Cyclophosphamide (6 µg/mL)	+	43.0	13.6	23	70	32

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test compound was a mixture of short- and long-chain acyl triglyceride.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted: 26 May 1983

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted: 29 Jul 2016

Deviations:

yes

Remarks:

only 100 cells per dose scored.

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary cells (CHO ETCC CCL 61 CHO-KL, proline-requiring)
- Type and identity of media: Cells were grown in a 5% C02 atmosphere at 37 "C in McCoy's 5a medium.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

250, 500 and 1000 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

yes

Remarks:

Corn oil; 500, 750 and 1000 µg/mL

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate , 20 µg/mL, -S9; cyclophosphamide, 110 µg/mL, +S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 8h (-S9); 2h (+S9);

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.4 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 50 cells per flask were evaluated for chromosomal aberrations, resulting in a total of 100 cells evaluated per dose.

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index was evaluated on the basis of at least 1000 cells per flask.

OTHER:
- In the absence of metabolic activation, the fats and 0.01 mM deoxybromouridine (BrdU) were added to the cell culture and incubated at 37ºC for 8h. The cells were washed and then incubated in fresh medium containing 0.01 mM BrdU for 13.5 h.
When metabolic activation was used, the cultures were exposed to the fats for 2 h at 37ºC, washed, and then incubated in fresh medium containing
0.01 mM BrdU for 6-8 h.
After incubation in BrdU, colchicine was added to the cultures at 0.4 μg/mL followed by incubation for 2.5 h at 37 ºC.

Evaluation criteria:

A test material would be considered positive if there was a statistically significant (p < 0.05) increase in the frequency of cells with chromosomal damage and if this increase was dose-dependent.

Statistics:

The number of cells with chromosomal damage in the test fat groups and positive control group were compared to the concurrent solvent control by Fischer's exact test (Gad and Weil, 1991).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The SALATRIM fat appeared to be soluble in the culture medium for the chromosomal aberration assay at a concentration of 40 μg/mL but produced a suspension at higher concentrations.
The pH of the high dose in the medium was approximately 7.0. The preliminary dose range/cytotoxicity study used a dose range of 0-1000 μg/mL and was limited by the lack of solubility of the fat. Corn oil behaved in a manner similar to that of SALATRIM 23CA lot A014 and was tested using the same criteria. No significant cell cycle delay or reduction in the mitotic index was noted with either corn oil or the SALATRIM fat. There was a slight increase in the number of cells in metaphase 1 at a SALATRIM dose of 1000 μg/mL and at corn oil doses of 40 and 200 μg/mL, but these were not significant on the basis of assay criteria. Therefore, for the definitive assay, a dose range of 0 (solvent control), 250, 500, and 1000 μg/mL was chosen for the SALATRIM and 500,750, and 1000 μg/mL for corn oil.

RANGE-FINDING/SCREENING STUDIES:
A preliminary dose range study to determine cytotoxicity and optimal cell fixation time was done before the definitive study was performed. Doses of 8.0, 40.0, 200.0, 500.0, and 1000 μg/mL were tested.
On the basis of the results of the range-finding study, doses of 250,500, and 1000 μg/mL for the SALATRIM fat and 500,750, and 1000 μg/mL for corn oil were used for the definitive study and a harvest time of 8-10 h was selected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of metabolic activation, no major changes were noted in the mitotic index with either SALATRIM 23CA lot A014 or corn oil, indicating a lack of cytotoxicity.

 

Table 1:Summary of results of the chromosome aberration study in CHO cells treated with SALATRIM 23CA Lot A014

 

	S9	Conc. in μg/mL	Cells scored	mitotic index (%)	cells with abnormal chromosomes (%)	cells with chromatid deletions /exchanges (%)	structural aberrations per cell
corn oil**	-	1000	100	9.6	2	2/0	0.02
MMS	-	20	100	4.7	18*	13/5	0.22
Acetone	-	0	100	5.9	4	3/0	0.04
Test item	-	250	100	4.8	2	1/0	0.01
Test item	-	500	100	5.0	4	3/0	0.06
Test item	-	1000	100	4.4	4	3/0	0.05
corn oil**	+	1000	100	4.7	5	4/0	0.05
CP	+	50	100	0.4	26*	16.2/6.1	0.42
Acetone	+	0	100	3.6	4	3/0	0.06
Test item	+	250	100	3.9	0	0/0	0.00
Test item	+	500	100	3.9	2	2/0	0.03
Test item	+	1000	100	5.1	3	3/0	0.03

*p<0.05

**corn oil was tested in an independent experiment

 

 

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted: 21 Jul 1997

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted: 29 Jul 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL

Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea (50 µg/mL, -S9), 7,12-dimethyl-1,2-benzanthracene (3.3 µg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)

- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period

When the above mentioned criteria were not met, the test item was considered as non-mutagenic.

Statistics:

Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid.

Table 1: First Experiment - 4 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.36	1
625	91	116	57	2.04	0.86
1250	108	94	65	2.44	1.03
2500	106	83	36	2.66	1.12
3600	89	76	11	0.30*	0.12
5000	74	83	8	0.04*	0.017
ENU (50)	53	62	45	#
Table 2: First Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.39	1
625	75	81	65	2.59	1.08
1250	92	44	46	3.04	1.27
2500	93	30	9	0.9	0.38
3600	90	5	1	0.2	0.08
5000	84	4	1	0.47	0.2
DMBA (3.3)	24	50	29	#
Table 3: Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	not reported	not reported
313	82	9	2	not reported	not reported
625	73	4	1	not reported	not reported
1250	68	4	1	not reported	not reported
2500	38	4	1	not reported	not reported
3600	31	1	0	not reported	not reported
ENU (50)	53	35	9	not reported	not reported
Table 4: Second Experiment - 3 h exposure - With Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2	1
156	83	95	62	1.95	0.98
313	84	101	70	1.84	0.92
625	82	105	65	1.79	0.9
1250	46	98.5	65	1.84	0.92
2500	38	88	37	1.93	0.97
3600	36	4	0	0	0
DMBA (3.3)	28	52	27	37.3	18.65
Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation
Concentration (µg/mL)	Cloning efficiency (%)		Relative Total Growth (%)	Mutants per 1E+4 surviving cells	Mutation factor
	Day 0	Day 3
0 (DMSO)	100	100	100	2.17	1
`2.5	102	100	62	2.16	1
5	100	114	151	2.02	0.93
10	92	95	145	1.97	0.91
20	105	100	60	2.11	0.97
40	82	95	103	2.17	1
80	69	111	97	1.82	0.84
160	25	44	16	2.32	1.07
320	11	31	6	1.94	0.89
ENU (50)	16	31	9	51.8	23.87

 

ENU: N-ethyl-N-nitrosourea

DMBA: 7,12-dimethyl-1,2-benzanthracene

*: statistically significant with p < 0.01

#: could not be calculated as all wells contained mutant colonies

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test compound was a mixture of short- and long-chain acyl triglyceride.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted: 4 Apr 1984

Deviations:

yes

Remarks:

exposure duration not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

29 Jul 2016

Deviations:

yes

Remarks:

exposure duration not reported

Principles of method if other than guideline:

The SALATRIM family of triacylglycerols differs from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA. These fats have caloric availability values (4.5-6 kcal/g) lower than that of corn oil (9 kcal/g). SALATRIM 23CA Lot
A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration, unscheduled DNA synthesis, and HPRT mammalian cell mutagenesis assays. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative
in the three assays.

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO) CHO-K1 cells obtained from the American Type Culture Collection
- Type and identity of media: in Ham’s F12 medium with 31 µg/mL penicillin, 50 µg/mL streptomycin sulfate, and 5 % heatinactivated
fetal bovine serum (F12/5).

Metabolic activation:

with and without

Metabolic activation system:

final S-9 concentration of 1 %

Test concentrations with justification for top dose:

0, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL
The high dose was limited by the low solubility of the fats in the assay medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
Stock solutions of SALATRIM 23CA lot A014 and corn oil were made in acetone such that the acetone never exceeded 1 % of the culture.

Untreated negative controls:

yes

Remarks:

Corn oil doses were 327.7-1000 μg/mL.

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Positive controls were EMS (200 μg/mL of culture) without metabolic activation and 3-MC (5 μg/mL of culture) with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: not reported
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14-17 days

Phenotypic expression of induced mutants was conducted by incubating the cells for 7 days. Cells were subcultured every 2-3 days by trypsinizing the flask, counting the cells, and replating 2 x 10(6) cells. After the expression period, approximately 3 x 10(6) cells from each culture were seeded in 100 mL of cloning medium supplemented with TG for selection of resistant cells. Approximately 600 cells were seeded in 100 mL of TG-free medium to determine the percentage of viable cells. The cells were incubated for 14-17 days and cell colonies counted with an ARTEK colony counter. Mutant frequency (ratio of mutant cells to nonmutant cells) was calculated by dividing the number of resistant colonies by the number of unselected viable colonies.

SELECTION AGENT (mutation assays): The selective cloning medium contained 30 µM 6-thioguanine (TG).

NUMBER OF REPLICATIONS: The definitive study was done in duplicate using duplicate cultures for each replicate.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by detaching the cells from the culture flask with 0.05% trypsin-0.02% EDTA. A Model 2F Coulter counter was used to determine cell numbers, and an aliquot containing at least 1-2 x 10(6) cells was added to 20 mL of F12/5 to determine phenotypic expression. The remaining cell suspension was diluted in approximately 35 mL of medium so that 500 cells were plated into two Petri dishes. After incubation for 14-17 days, cell colonies were determined. Survival was expressed as the cloning efficiency (CE) relative to the solvent control.

Evaluation criteria:

The test results were considered positive if a dose-related increase in the number of mutant colonies occurred and the mutant frequencies of duplicate cultures treated with one or more concentrations of the test article were at least 3 times the average of those from the solvent control cultures.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Although the test material was soluble in the acetone, turbidity was noted in the cultures, indicating solubility had been exceeded.

RANGE-FINDING/SCREENING STUDIES:
Preliminary experiments were done to determine cytotoxicity and aid in selection of the dose range.

Table 3: Results of the HPRT Mammalian Cell Gene Mutation Assay of SALATRIM 23CA lot A014

with and without Metabolic Activation*

SALATRIM Dose (μg/mL)	without metabolic activation		with metabolic activation
	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)	rel. cloning efficiency (% of control)	mutant frequency (per 10^6cells)
0	100	15	100	10
31.3	96	7	94	7
62.5	100	18	84	6
125	89	11	93	10
250	90	17	97	15
500	78	12	69	9
1000	86	17	75	10
corn oil 1000**	91	6	79	15
EMS200	56	170	-	-
3MC 5	-		79	170

 

 

*Data represent the mean of two cultures with the exception of the 0 dose (solvent control), which represents the mean of triplicate cultures.

**Data from a independent experiment with corn oil as test substance instead of SALATRIM

EMS= Ethyl methanesulfonate; positive control for the assay without metabolic activation.

3MC = 3-Methylcholanthrene; positive control for the assay with metabolic activation.

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across

There is data on the genetic toxicity in bacteria in vitro for Dodecanoic acid, ester with 1,2,3-propanetriol (CAS 37318-95-9). There is no data available on the in vitro cytogenicity and gene mutation in mammalian cells of the target substance. The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 37318-95-9 (target)

In a GLP-study performed according to OECD guideline 471, Dodecanoic acid, ester with 1,2,3-propanetriol (CAS 37318-95-9)was investigated in S. typhimurium TA 1535, TA102, TA 100, TA 98 and TA97a (WoE, 2018). In two dose finding tests with test concentrations up to 15000 µg/plate, suitable concentrations for treatment of the bacterial strains in the main study were selected. Based on the results obtained in the pre-test, concentrations up to 15 µg/plate were chosen in the absence and presence of metabolic activation. Cytotoxic effects indicated by a decrease in spontaneous revertants were observed at 15 µg/plate in all bacterial strains with and without metabolic activation. No precipitation was noted at any concentration tested. No increase in the mean number of revertants per plate was observed in any strain compared to controls. The positive and negative controls included in the assay demonstrated the validity of the assay. Under the conditions of this assay, the test substance was not mutagenic in the presence or absence of metabolic activation.

 

Cytogenicity in mammalian cells in vitro

CAS 91052-13-0

The clastogenic potential of Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was assessed in a GLP-study in Chinese hamster lung cells (CHL/IU) according to OECD guideline 473 (WoE, 2010). In a preliminary cell growth inhibition test with concentrations ranging from 9.8 to 5000 µg/plate, no significant inhibition of cell growth was observed after 4 h exposure in the presence and absence of metabolic activation (S9 mix). However, a moderate reduction of cell growth of ca. 30-40% compared to controls was observed at concentrations ranging from 156-1250 µg/mL after 24 and 48 h continuous exposure. Based on the results of this study, concentrations of 20, 39, 78 and 156 µg/mL (± S9 mix) were used for the analysis of chromosomal aberrations after short-term exposure (6 h), whereas concentrations of 78, 156, 313, 625, 1250, 2500 and 5000 µg/mL (without S9 mix) were chosen for the continuous exposure treatment (24 and 48 h). Per culture, 100 cells were scored. No increase in the number of cells with chromosomal aberrations was observed compared with controls in any of the experiments performed. No cytotoxic effects were observed in any of the experiments performed. An oily precipitation of the test substance was observed at concentrations ≥ 78 µg/mL, but did not interfere with the chromosomal analysis of the treated cells. The positive controls included during short-term and continuous exposure showed the expected results. Under the conditions of this experiment, the test substance was considered to be not clastogenic in Chinese hamster lung cells (CHL/IU) in the presence and absence of metabolic activation.

CAS 736150-63-3

An in vitro mammalian chromosome aberration test was performed with Glycerides, castor-oil mono, hydrogenated, acetates in human lymphocytes according to OECD guideline 473 and in compliance with GLP (WoE, 2004). An initial cytotoxicity test was performed with concentrations of 313, 625, 1250, 2500 and 5000 µg/mL in the presence or absence of metabolic activation (S9-mix) for of 3 h. A dose-related toxicity was observed. At the highest concentration (5000 µg/mL), the mitotic index was reduced to 64% of the vehicle control without metabolic activation and to 10% with metabolic activation, respectively. Based on these results, concentrations of 625, 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250, 2500, 3600 and 5000 µg/mL (with 2% S9-mix) were chosen for treatment of cells in the main assay for an exposure period of 3 h. Metaphase analysis was performed at concentrations of 1250, 2500 and 5000 µg/mL (without S9-mix) and 625, 1250 and 2500 µg/mL (with 2% S9-mix). In the second main assay, cells were exposed to concentrations of 313-5000 µg/mL (without S9 mix) or 625-5000 µg/mL (with 4% S9 mix) for either 3 or 20 h, respectively. As the samples without metabolic activation revealed mean mitotic indices lower than 50% of the solvent control in all dose groups, this part of the experiment was repeated with lower concentrations ranging from 2.5 to 320 µg/mL. Based on the cytotoxicity data obtained, concentrations of 40, 80 and 160 µg/mL (without S9) and 625, 1250 and 2500 µg/mL (with 4% S9-mix) were used for metaphase analysis. At least 100 cells were scored per culture. No treatment-related increase in the number of cells with chromosomal aberrations compared with the controls was observed with or without metabolic activation for any concentration level. The frequency of metaphases with chromosomal aberrations in the solvent controls was compatible to the historical control values and the positive controls produced statistically significant increases in the frequency of metaphases with chromosomal aberrations. Based on the results of this chromosome aberration test, the test substance was not clastogenic in human lymphocytes in the presence or absence of metabolic activation under the experimental conditions chosen.

Short-, medium- and long-chain triglycerides (SCT, MCT, LCT)

An in vitro mammalian chromosome aberration test with the SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols was performed similar to OECD guideline 473 in Chinese hamster Ovary (CHO) cells (WoE, 1994). In a preliminary toxicity test, concentrations of 8.0, 40.0, 200.0, 500.0, and 1000 μg/mL were used to determine suitable concentrations for chromosome analysis. Based on these results, the incidence of chromosome aberrations was assessed in the presence and absence of metabolic activation (rat liver S9-mix) with test substance concentrations of 250, 500 and 1000 µg/mL. The exposure duration was 2 h with metabolic activation, and 8 h without metabolic activation. The high dose was selected based on the low solubility of the fats in the assay medium. A total of 100 cells were scored per culture. No significant increase in the number of phases with aberrations was observed in treated CHO cells at any preparation time and concentration. No significant cytotoxic effects were reported. The positive controls significantly increased the rate of chromosome aberrations. In conclusion, the test substance was not clastogenic in Chinese hamster ovary cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions chosen.

Mutagenicity in mammalian cells in vitro

CAS 736150-63-3

An in vitro mammalian cell gene mutation assay was performed with Glycerides, castor-oil mono, hydrogenated, acetates according to OECD guideline 476 and under GLP conditions (WoE, 2002). In the first experiment, mutations at the TK locusof mouse-lymphoma L5178Y cells were investigated at concentrations of 625, 1250, 2500, 3600 and 5000 µg/mL. In this experiment, cells were exposed to the test material for a period of 3 h in the presence and for 4 h in the absence of metabolic activation (S9-mix). The fixation time was 13 – 14 days. At 3600 µg/mL, the relative total growth was 10-20% compared to the negative controls, thus providing an appropriate maximum concentration for treatment in the second experiment. In this experiment, cells were exposed without S9-mix to concentrations ranging from 313 to 3600 µg/ for a period of 24 h, whereas in the presence of S9-mix, cells were treated with concentrations of 156-3600 µg/mL for a period of 4 h. Since the relative growth with S9-mix was very low (0-2%) at all test concentrations, the 24-h treatment of cells in the absence of S9-mix was repeated with concentrations ranging from 2.5-320 µg/mL, which resulted in appropriate levels of cytotoxicity (10-20% relative growth) at 160 µg/mL. In the presence of metabolic activation, the relative total growth was 9% at 2500 µg/mL in the first experiment and 37 and 0% at 2500 and 3600 µg/mL in the second experiment, respectively. After a 3-day expression period of the cultures, the resistance to 5-trifluorothymidine (TFT) was determined in all experiments. The test substance did not induce a significant increase in the mutant frequency at any preparation time and dose concentration. The positive controls significantly increased mutant frequency. In conclusion, the test substance did not induce mutations in mouse-lymphoma L5178Y cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions.

Short-, medium- and long-chain triglycerides (SCT, MCT, LCT)

The SALATRIM (short- and long-chain acyl triglyceride molecules) family of triacylglycerols was assessed using a gene mutation assay in cultured mammalian cells (Chinese hamster ovary (CHO-K1) cells) similarly to OECD guideline 476 (WoE, 1994). Gene mutations at the HPRT locus were investigated in the presence and absence of metabolic activation (rat liver S9-mix) with concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/mL. The highest concentration was limited by the low solubility of the fats in the assay medium. The expression time was 7 days and the selection time was 14 – 17 days. No significant cytotoxicity was reported. An increase in mutant frequency was not observed at any concentration tested whether with or without metabolic activation. The positive controls significantly increased the mutant frequency. Therefore, it was concluded that under the conditions of the study, the test material was not mutagenic at the HPRT locus of Chinese hamster ovary cells in the absence and presence of metabolic activation.

 

Overall conclusion for genetic toxicity

For the target substance Dodecanoic acid, ester with 1,2,3-propanetriol, the Ames test (in vitro genetic toxicity in bacteria) was negative. To meet the requirements for the genetic toxicity endpoint, analogue read-across from 3 source substances was applied fromin vitro studies on cytogenicity and genetic toxicity in mammalian cells. The results of the available studies were consistently negative. Based on the available data on the target and source substances,Dodecanoic acid, ester with 1,2,3-propanetriol is not expected to be clastogenic or mutagenic.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Dodecanoic acid, ester with 1,2,3-propanetriol, data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the available data on analogue read-across approach, the results on genetic toxicity for the source substances do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.