Decanoic acid, monoester with glycerol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May - 07 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Esterification product of glycerol and C8-C12 (even numbered) fatty acids

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Esterification product of glycerol and C8-C12 (even numbered) fatty acids

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Exp 1a: 5000, 1500, 500, 150 and 50 µg/plate for all strains with and without metabolic activation
Exp 1b: 1500, 500, 150, 50 and 5 µg/plate for TA97a, TA98, TA100 and TA102 with and without metabolic activation
Exp 1b: 500, 150, 50, 15,5 and 1.5 µg/plate for TA1535

Exp 2a: 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 µg/plate for TA97a, TA98, TA100 and TA102 with and without metabolic activation
Exp 2a: 150, 75, 37.5, 18.8, 9.4, 4.7 and 2.3 µg/plate for TA1535 with and without metabolic activation
Exp 2b: 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 µg/plate for TA98 with and without metabolic activation

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and deionised water

Positive control substance:

sodium azide

benzo(a)pyrene

other: see "remarks"

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Exp 1a and b); preincubation (Exp 2a and b)

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

NUMBER OF REPLICATIONS: 3 replications each in 3 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the 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 deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1 and 2: at 500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1 and 2: at 1500 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item showed no precipitates on the plates at any of the concentrations.
- Contamination: Due to the contamination of the bacteria strain TA98, a repetition of the experiment 2a for the bacteria strain TA98 (Exp 2b) was performed.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item at 5000 μg/plate was tested in a preliminary experiment at strains TA97a, TA98, TA100, TA102 and TA1535. Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar for 48 hours at 37 ±1°C. Toxicity was observed in all strains at 5000 µg/plate.

HISTORICAL CONTROL
All negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 1. Mean number of revertant colonies - Experiment 1a (plate incorporation method)

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	73	82	12	11	77	75	273	313	14	17
	SD	10.6	6.5	2.6	1.5	8.1	11.0	14.0	22.0	3.1	3.5
DMSO	Mean	80	90	9	11	80	85	276	256	22	19
	SD	15.0	13.1	1.2	1.5	5.5	25.6	17.4	10.6	2.1	4.0
Positive Controls*	Mean	337	404	496	74	284	361	719	769	309	113
	SD	41.1	77.9	39.4	14.0	16.0	18.5	18.0	103.9	59.0	23.2
	f(I)	4.21	4.49	55.11	6.73	3.69	4.25	2.61	3.00	22.07	5.95
5000 µg/plate	Mean	0	0	0	0	0	0	30	40	0	0
	SD	0.0	0.0	0.0	0.0	0.0	0.0	8.3	15.7	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.11	0.16	0.00	0.00
1500 µg/plate	Mean	0	0	0	0	4	27	156	220	0	0
	SD	0.0	0.0	0.0	0.0	0.0	11.8	12.5	55.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.05	0.32	0.57	0.86	0.00	0.00
500 µg/plate	Mean	77	79	10	9	73	71	309	255	4	9
	SD	9.5	21.9	0.0	1.0	9.2	8.2	37.2	38.4	2.9	2.9
	f(I)	0.96	0.88	1.11	0.82	0.91	0.84	1.12	1.00	0.18	0.47
150 µg/plate	Mean	79	76	12	9	84	75	240	281	19	26
	SD	5.1	6.1	1.0	4.0	6.1	13.2	45.1	42.8	4.0	3.8
	f(I)	0.99	0.84	1.33	0.82	1.05	0.88	0.87	1.10	0.86	1.37
50 µg/plate	Mean	101	75	13	17	79	73	257	299	24	27
	SD	13.4	5.5	4.0	2.5	5.2	15.9	30.3	31.1	2.6	6.0
	f(I)	1.26	0.83	1.44	1.55	0.99	0.86	0.93	1.17	1.09	1.42

f(l): increase factor (mean revertants divided by mean spontaneous revertants)

*Different positive controls were used, see “controls”

Table 2. Mean number of revertant colonies- Experiment 1b (plate incorporation method)

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	74	77	18	20	79	94	307	327	16	13
	SD	8.9	14.6	4.0	1.2	13.1	8.5	29.5	66.0	0.6	1.5
DMSO	Mean	75	71	25	23	71	80	288	328	14	14
	SD	7.8	10.1	4.5	4.7	7.1	8.4	41.8	72.8	2.1	2.0
Positive Controls*	Mean	322	454	224	195	481	412	2235	1355	216	143
	SD	36.7	154.2	36.0	12.2	9.2	14.4	68.0	382.5	25.1	43.9
	f(I)	4.29	6.39	8.96	8.48	6.09	5.15	7.76	4.13	13.50	10.21
1500 µg/plate	Mean	0	0	0	0	0	0	0	0	n.d.	n.d.
	SD	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	n.d.	n.d.
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	n.d.	n.d.
500 µg/plate	Mean	96	65	19	23	70	73	206	237	0	0
	SD	12.9	6.0	2.0	3.5	3.6	19.1	51.0	72.6	0.0	0.0
	f(I)	1.28	0.92	0.76	1.00	0.99	0.91	0.72	0.72	0.00	0.00
150 µg/plate	Mean	71	88	23	21	69	75	297	358	14	11
	SD	10.8	14.6	1.5	1.2	17.2	4.0	30.0	83.2	3.8	3.2
	f(I)	0.95	1.24	0.92	0.91	0.97	0.94	1.03	1.09	1.00	0.79
50 µg/plate	Mean	92	91	22	22	78	68	341	341	15	12
	SD	15.6	21.5	1.0	2.1	19.9	6.1	98.8	32.1	3.5	4.0
	f(I)	1.23	1.28	0.88	0.96	1.10	0.85	1.18	1.04	1.07	0.86
15 µg/plate	Mean	79	103	22	22	68	74	231	229	14	12
	SD	7.6	10.5	4.0	5.6	7.6	9.5	66.6	34.0	3.1	3.5
	f(I)	1.05	1.45	0.88	0.96	0.96	0.93	0.80	0.70	1.00	0.86
5 µg/plate	Mean	74	101	23	23	83	79	284	296	14	16
	SD	17.1	15.4	3.2	2.5	6.7	12.5	42.1	52.9	3.2	0.6
	f(I)	0.99	1.42	0.92	1.00	1.17	0.99	0.99	0.90	1.00	1.14
1.5 µg/plate	Mean	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	13	14
	SD	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	1.0	2.5
	f(I)	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.93	1.00

 f(l): increase factor (mean revertants divided by mean spontaneous revertants)

*Different positive controls were used, see “controls”

n.d.: not determined

Table 3a. Mean number of revertant colonies - Experiment 2a (preincubation method)

Strain		TA97a		TA98		TA100		TA102
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	79	105	n.e.	n.e.	76	87	349	325
	SD	14.2	10.8	n.e.	n.e.	10.7	3.5	18.9	40.1
DMSO	Mean	83	114	n.e.	n.e.	80	75	340	340
	SD	16.9	5.5	n.e.	n.e.	4.0	5.3	46.1	18.3
Positive Controls*	Mean	489	379	n.e.	n.e.	312	755	1084	1135
	SD	25.8	88.1	n.e.	n.e.	36.7	116.6	17.4	68.9
	f(I)	5.89	3.32	n.e.	n.e.	4.11	10.07	3.19	3.34
500 µg/plate	Mean	0	51	n.e.	n.e.	0	43	267	333
	SD	0.0	8.5	n.e.	n.e.	0.0	11.1	19.7	8.3
	f(I)	0.00	0.45	n.e.	n.e.	0.00	0.57	0.79	0.98
250 µg/plate	Mean	73	82	n.e.	n.e.	26	79	281	281
	SD	7.5	14.6	n.e.	n.e.	8.5	9.0	31.1	18.0
	f(I)	0.88	0.72	n.e.	n.e.	0.33	1.05	0.83	0.83
125 µg/plate	Mean	71	101	n.e.	n.e.	67	72	271	300
	SD	5.9	9.1	n.e.	n.e.	5.6	1.5	26.6	38.2
	f(I)	0.86	0.89	n.e.	n.e.	0.84	0.96	0.80	0.88
62.5 µg/plate	Mean	75	87	n.e.	n.e.	74	75	279	299
	SD	6.0	16.7	n.e.	n.e.	6.6	7.6	14.0	4.6
	f(I)	0.90	0.76	n.e.	n.e.	0.93	1.00	0.82	0.88
31.3 µg/plate	Mean	72	84	n.e.	n.e.	81	79	337	313
	SD	4.7	12.2	n.e.	n.e.	7.9	5.5	25.7	33.3
	f(I)	0.87	0.74	n.e.	n.e.	1.01	1.05	0.99	0.92
15.6 µg/plate	Mean	72	77	n.e.	n.e.	72	80	309	305
	SD	9.5	14.0	n.e.	n.e.	7.5	13.6	12.2	19.7
	f(I)	0.87	0.68	n.e.	n.e.	0.90	1.07	0.91	0.90
7.8 µg/plate	Mean	75	75	n.e.	n.e.	80	74	265	353
	SD	5.5	9.5	n.e.	n.e.	2.6	5.0	20.1	64.8
	f(I)	0.90	0.66	n.e.	n.e.	1.00	0.99	0.78	1.04

n.e.: not evaluated, due to contamination

f(l): increase factor (mean revertants divided by mean spontaneous revertants)

*Different positive controls were used, see “controls”

Table 3b. Mean number of revertant colonies of TA1535 - Experiment 2a (preincubation method)

Strain		TA1535
Induction		-S9	+S9
Demin. water	Mean	22	18
	SD	3.2	2.6
DMSO	Mean	20	21
	SD	4.0	2.0
Positive Controls*	Mean	277	77
	SD	48.9	8.6
	f(I)	12.59	3.67
150 µg/plate	Mean	13	21
	SD	1.5	3.5
	f(I)	0.65	1.00
75 µg/plate	Mean	20	24
	SD	7.2	4.9
	f(I)	1.00	1.14
37.5 µg/plate	Mean	19	21
	SD	4.7	4.0
	f(I)	0.95	1.00
18.8 µg/plate	Mean	20	21
	SD	3.6	2.1
	f(I)	1.00	1.00
9.4 µg/plate	Mean	21	18
	SD	2.5	2.5
	f(I)	1.05	0.86
4.7 µg/plate	Mean	20	20
	SD	3.8	5.1
	f(I)	1.00	0.95
2.3 µg/plate	Mean	18	19
	SD	4.5	3.8
	f(I)	0.90	0.90

f(l): increase factor (mean revertants divided by mean spontaneous revertants)

*Different positive controls were used, see “controls”

Table 4. Mean number of revertant colonies of TA98 - Experiment 2b (preincubation method)

Strain		TA98
Induction		-S9	+S9
Demin. water	Mean	17	17
	SD	1.0	1.0
DMSO	Mean	16	14
	SD	2.6	0.0
Positive Controls*	Mean	331	106
	SD	34.0	14.6
	f(I)	19.47	7.57
500 µg/plate	Mean	0	0
	SD	0.0	0.0
	f(I)	0.00	0.00
250 µg/plate	Mean	2	3
	SD	1.2	0.0
	f(I)	0.11	0.18
125 µg/plate	Mean	12	13
	SD	2.6	1.5
	f(I)	0.63	0.76
62.5 µg/plate	Mean	14	11
	SD	0.0	2.0
	f(I)	0.74	0.65
31.3 µg/plate	Mean	13	11
	SD	1.5	0.6
	f(I)	0.68	0.65
15.6 µg/plate	Mean	13	13
	SD	0.6	0.6
	f(I)	0.68	0.76
7.8 µg/plate	Mean	15	15
	SD	1.0	4.0
	f(I)	0.79	0.88

f(l): increase factor (mean revertants divided by mean spontaneous revertants)

*Different positive controls were used, see “controls”

Applicant's summary and conclusion

Conclusions:

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