Ethyl decanoate

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:

From 10 November 2017 to 15 December 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl decanoate

EC Number:

203-761-9

EC Name:

Ethyl decanoate

Cas Number:

110-38-3

Molecular formula:

C12H24O2

IUPAC Name:

ethyl decanoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 16090585
- Expiration date of the lot/batch: 19.09.2018
- Purity test date: 22.09.2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: soluble at 5µL/plate
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was diluted with the vehicle
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: the test item was diluted with the vehicle for the high dose concentration used and the other concentrations were prepared by serial dilution 1/3
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Test item diluted in vehicle

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa mutation, uvrB deletion (S. typhimurium), uvrA deletion (WP2 pKM101)

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial supernatant( S9) of liver from rats which were treated with Aroclor 1254 intravenously

Test concentrations with justification for top dose:

The test item was soluble in the assay final mixture (Corn oil with PBS) at a concentration of 5 μL/plate, with no precipitation signs being observed in the assay final mixture with PBS. On the basis of the solubility and cytotoxicity results of the test item, the C5 selected for the main test was 5 μL/plate. Concentrations C4 to C1 (C4: 1.667 µL/plate; C3: 0.556 µIL/plate ; C2 : 0.158 µL/plate; C1: 0.062 µL/plate) were prepared by 1:3 serial dilutions in the selected solvent from the C5 concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: corn oil with PBS
- Justification for choice of solvent/vehicle:The test item was soluble in the assay final mixture (Corn oil with PBS) at a concentration of 5 μL/plate, with no precipitation signs being observed in the assay final mixture with PBS.
Therefore, the C5 selected for the cytotoxicity assay was 5 μL/plate.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicates were used

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: the revertant colonies were counted by an automatic colony counter

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity evaluation of the test item was based on the decrease in the number of revertant colonies, or a clearing or diminution of the background lawn.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity evaluation of the test item was performed in the S. typhimurium TA100 strain by the direct incorporation procedure and without metabolic activation (S9) using 5 concentrations based on the solubility profile of the test item which ranged from 0,062 up to 5,00 μL/plate. Test item solutions were prepared by 1:3 serial dilution of C5.

Rationale for test conditions:

The Ames test evaluates the potential of the test item to revert mutations present in amino acid-requiring bacterial strains. The reversion restores the functional capability of the bacteria to synthesize the essential amino acid thus enabling the bacterial culture to grow in the absence of the amino acid required by the parent bacterial strain. The mutagenic or pro-mutagenic potential of the test item is assessed by the increase in the number of revertant colonies upon exposure to the test item relative to the number of spontaneously occurring revertant colonies in the controls.

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result is considered positive whenever the number of revertants of the test item-treated plates is increased when compared to the solvent-treated plates (2 fold increase for TA98, TA 100 and WP2 and 3 fold increase for TA 1537 and TA1535)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : see attached table in section "Any other information on results incl. tables"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:No test item related cytotoxic activity was observed in the bacterial system over the concentration range tested of 0,062 up to 5,00 μL/plate.

Any other information on results incl. tables

 HISTORICAL CONTROLVALUES

 

 

	Metabolic								Reference interval (mean ± 2.58SD)
Strain	activation	Control		Mean	SD	Max	Min	n	Max	Min
TA98	(-S9)	d.i.	+	430	71.88	633	306	219	616	245
			-	22	4.86	42	13	255	35	10
		p.i.	+	414	69.17	627	299	219	592	235
			-	22	4.91	48	12	255	35	10
	(+S9)	d.i.	+	687	91.94	920	357	219	924	450
			-	24	5.67	52	14	258	38	9
		p.i.	+	636	94.94	872	382	219	881	392
			-	24	5.53	45	14	258	38	9
TA100	(-S9)	d.i.	+	825	79.69	1009	620	219	1031	619
			-	87	15.69	153	46	255	128	47
		p.i.	+	828	82.76	1087	658	219	1041	614
			-	89	14.78	153	61	255	127	50
	(+S9)	d.i.	+	1615	191.90	1983	1039	219	2110	1120
			-	93	16.77	147	61	258	136	49
		p.i.	+	1454	211.56	1977	976	219	2000	908
			-	92	15.81	135	62	258	133	51
TA102	(-S9)	d.i.	+	1082	130.79	1347	939	24	1419	744
			-	388	49.57	483	304	24	516	260
		p.i.	+	1198	164.85	1590	928	24	1624	773
			-	393	39.04	470	313	24	494	293
	(+S9)	d.i.	+	2037	182.44	2326	1687	24	2507	1566
			-	387	49.46	475	288	24	515	259
		p.i.	+	2028	238.25	2667	1682	24	2643	1414
			-	366	35.93	436	291	24	459	273
TA1535	(-S9)	d.i.	+	892	93.07	1162	602	216	1132	652
			-	19	5.10	31	6	252	32	6
		p.i.	+	921	88.41	1167	603	216	1149	693
			-	19	4.97	37	9	252	32	6
	(+S9)	d.i.	+	358	67.32	566	122	216	532	185
			-	19	5.32	34	9	255	32	5
		p.i.	+	391	71.60	556	165	216	576	206
			-	19	5.10	32	5	255	32	6
TA1537	(-S9)	d.i.	+	187	27.26	346	121	219	257	116
			-	6	1.90	12	2	255	11	1
		p.i.	+	183	25.76	293	123	219	250	117
			-	6	1.85	10	2	255	11	1
	(+S9)	d.i.	+	196	28.79	295	130	219	271	122
			-	6	2.07	12	2	258	12	1
		p.i.	+	182	30.28	299	105	219	260	104
			-	6	2.03	11	2	258	12	1
WP2	(-S9)	d.i.	+	1941	168.46	2315	1313	213	2376	1506
			-	244	40.71	351	144	249	349	139
		p.i.	+	2092	196.82	2759	1345	213	2600	1584
			-	245	40.53	358	160	249	349	140
	(+S9)	d.i.	+	2023	175.00	2985	1627	213	2475	1572
			-	256	48.07	381	117	252	380	132
		p.i.	+	2011	149.25	2332	1602	213	2396	1626
			-	258	45.45	412	156	252	375	141

d.i.: direct incorporation /p.i.: pre-incubation/ +:referencecontrol / -:solventcontrol / n:number of values

 

 

RESULTSOFTHECYTOTOXICITYASSAY                                                                                                          

S.typhimurium         TA100

			amount/plate	revertants/plate		mean            SD	R
Solvent:	Corn oil		‐	125              122	102	116.3          12.5	‐
			5.000	96               109	87	97.3           11.1	0.8
			1.667	104               94	107	101.7           6.8	0.9
	Test item	µL	0.556	114              108	125	115.7           8.6	1.0
			0.185	108              108	110	108.7           1.2	0.9
			0.062	118              103	105	108.7           8.1	0.9

 

RESULTS OF THE TEST WITHOUT METABOLIC ACTIVATION/DIRECT INCORPORATION PROCEDURE

 

TA98

 

 

 

amount/plate		revertants/ plate			mean	SD	R
Solvent:        Corn oil	‐	18	31	20	23.0	7.0	‐
Reference item (µg):  2‐nitrofluorene	5	417	395	577	463.0	99.3	20.1
	5.000	12	28	23	21.0	8.2	0.9
	1.667	26	23	25	24.7	1.5	1.1
Test item / µL	0.556	19	18	18	18.3	0.6	0.8
	0.185	13	26	23	20.7	6.8	0.9
	0.062	19	27	28	24.7	4.9	1.1

  

 

TA100

Solvent:	Corn oil	‐	108	102	98	102.7	5.0	‐
Referenceitem(µg):	sodiumazide	2.5	947	1000	983	976.7	27.1	9.5
		5.000	110	117	97	108.0	10.1	1.1
		1.667	118	121	107	115.3	7.4	1.1
Test item µL		0.556	127	105	96	109.3	15.9	1.1
		0.185	134	138	109	127.0	15.7	1.2
		0.062	110	131	119	120.0	10.5	1.2

 

TA1535

Solvent:	Corn oil	‐	19	35	20	24.7	9.0	‐
Referenceitem(µg):	sodiumazide	3.5	1062	1091	1042	1065.0	24.6	43.2
		5.000	26	17	20	21.0	4.6	0.9
		1.667	18	27	18	21.0	5.2	0.9
Test item µL		0.556	19	16	20	18.3	2.1	0.7
		0.185	15	10	28	17.7	9.3	0.7
		0.062	15	20	18	17.7	2.5	0.7

 

TA1537

Solvent:	Corn oil	‐	9	8	8	8.3	0.6	‐
Referenceitem(µg):	9‐aminoacridine	45	168	181	130	159.7	26.5	19.2
		5.000	6	8	4	6.0	2.0	0.7
		1.667	13	11	9	11.0	2.0	1.3
Test item µL		0.556	10	9	4	7.7	3.2	0.9
		0.185	10	13	7	10.0	3.0	1.2
		0.062	7	9	8	8.0	1.0	1.0

 

WP2

Solvent:	Cornoil	‐	257	292	240	263.0	26.5	‐
Referenceitem(µg):	4‐nitroquinoline‐N‐oxide	0.4	2050	1717	1850	1872.3	167.6	7.1
		5.000	291	289	226	268.7	37.0	1.0
		1.667	338	292	242	290.7	48.0	1.1
Test item µL		0.556	294	295	227	272.0	39.0	1.0
		0.185	286	319	228	277.7	46.1	1.1
		0.062	255	289	201	248.3	44.4	0.9

RESULTS OF THE TEST WITHOUT METABOLIC ACTIVATION/PRE‐INCUBATION PROCEDURE

 

TA98

amount/plate		revertants/plate			mean	SD	R
Solvent:                   Cornoil	‐	23	22	14	19.7	4.9	‐
Referenceitem(µg):  2‐nitrofluorene	5	450	477	469	465.3	13.9	23.7
	5.000	21	19	11	17.0	5.3	0.9
	1.667	20	20	13	17.7	4.0	0.9
TestitemµL	0.556	18	15	16	16.3	1.5	0.8
	0.185	22	25	21	22.7	2.1	1.2
	0.062	13	13	15	13.7	1.2	0.7

TA100

Solvent:	Cornoil	‐	92	123	91	102.0	18.2	‐
Referenceitem(µg):	sodiumazide	2.5	876	920	830	875.3	45.0	8.6
		5.000	99	118	102	106.3	10.2	1.0
		1.667	117	125	114	118.7	5.7	1.2
Test item µL		0.556	125	135	106	122.0	14.7	1.2
		0.185	126	110	113	116.3	8.5	1.1
		0.062	121	135	111	122.3	12.1	1.2

 

TA1535

Solvent:	Cornoil	‐	16	18	13	15.7	2.5	‐
Referenceitem(µg):	sodiumazide	3.5	827	926	869	874.0	49.7	55.8
		5.000	19	11	14	14.7	4.0	0.9
		1.667	16	14	10	13.3	3.1	0.9
Test item µL		0.556	18	20	10	16.0	5.3	1.0
		0.185	13	13	12	12.7	0.6	0.8
		0.062	12	11	16	13.0	2.6	0.8

 

TA1537

Solvent:	Cornoil	‐	9	8	10	9.0	1.0	‐
Referenceitem(µg):	9‐aminoacridine	45	192	203	149	181.3	28.5	20.1
		5.000	7	11	6	8.0	2.6	0.9
		1.667	11	5	4	6.7	3.8	0.7
Test item µL		0.556	8	2	3	4.3	3.2	0.5
		0.185	7	9	9	8.3	1.2	0.9
		0.062	7	7	7	7.0	0.0	0.8

 

WP2

Solvent:	Cornoil	‐	255	298	246	266.3	27.8	‐
Referenceitem(µg):	4‐nitroquinoline‐N‐oxide	0.4	1999	2181	2018	2066.0	100.0	7.8
		5.000	336	361	267	321.3	48.7	1.2
		1.667	287	316	290	297.7	15.9	1.1
Test item µL		0.556	349	289	340	326.0	32.4	1.2
		0.185	336	269	299	301.3	33.6	1.1
		0.062	330	318	282	310.0	25.0	1.2

RESULTS OF THE TEST WITH METABOLIC ACTIVATION/DIRECT INCORPORATION PROCEDURE

 

 

TA98

amount/plate		revertants/plate			mean	SD	R
Solvent:                   Cornoil	‐	34	22	25	27.0	6.2	‐
Referenceitem(µg):  2‐amino‐anthracene	1.5	695	555	596	615.3	72.0	22.8
	5.000	15	12	17	14.7	2.5	0.5
	1.667	33	35	23	30.3	6.4	1.1
TestitemµL	0.556	24	19	24	22.3	2.9	0.8
	0.185	23	24	30	25.7	3.8	1.0
	0.062	17	29	18	21.3	6.7	0.8

TA100

Solvent:	Cornoil	‐	118	127	110	118.3	8.5	‐
Referenceitem(µg):	2‐amino‐anthracene	2.5	1884	1759	1723	1788.7	84.5	15.1
		5.000	95	117	102	104.7	11.2	0.9
		1.667	109	143	106	119.3	20.6	1.0
TestitemµL		0.556	131	111	141	127.7	15.3	1.1
		0.185	118	123	128	123.0	5.0	1.0
		0.062	127	104	104	111.7	13.3	0.9

 

TA1535

Solvent:	Cornoil	‐	22	22	24	22.7	1.2	‐
Referenceitem(µg):	2‐amino‐anthracene	30	402	366	338	368.7	32.1	16.3
		5.000	12	17	8	12.3	4.5	0.5
		1.667	15	19	15	16.3	2.3	0.7
TestitemµL		0.556	16	22	16	18.0	3.5	0.8
		0.185	16	19	19	18.0	1.7	0.8
		0.062	31	13	13	19.0	10.4	0.8

 

TA1537

Solvent:	Cornoil	‐	8	8	10	8.7	1.2	‐
Referenceitem(µg):	2‐amino‐anthracene	2.5	186	216	198	200.0	15.1	23.1
		5.000	6	4	12	7.3	4.2	0.8
		1.667	4	10	9	7.7	3.2	0.9
TestitemµL		0.556	13	7	7	9.0	3.5	1.0
		0.185	8	13	2	7.7	5.5	0.9
		0.062	12	6	6	8.0	3.5	0.9

 

WP2

Solvent:	Cornoil	‐	241	266	305	270.7	32.3	‐
Referenceitem(µg):	2‐amino‐anthracene	30	1870	1620	1757	1749.0	125.2	6.5
		5.000	308	247	259	271.3	32.3	1.0
		1.667	309	382	221	304.0	80.6	1.1
TestitemµL		0.556	361	335	302	332.7	29.6	1.2
		0.185	337	378	301	338.7	38.5	1.3
		0.062	253	249	200	234.0	29.5	0.9

RESULTS OF THE TEST WITH METABOLIC ACTIVATION/PRE‐INCUBATION PROCEDURE

 

 

TA98

amount/plate		revertants/plate			mean	SD	R
Solvent:                   Cornoil	‐	29	15	19	21.0	7.2	‐
Referenceitem(µg):  2‐amino‐anthracene	1.5	811	639	703	717.7	86.9	34.2
	5.000	29	13	29	23.7	9.2	1.1
	1.667	28	15	26	23.0	7.0	1.1
TestitemµL	0.556	32	28	27	29.0	2.6	1.4
	0.185	27	28	21	25.3	3.8	1.2
	0.062	26	22	23	23.7	2.1	1.1

TA100

Solvent:	Cornoil	‐	128	106	114	116.0	11.1	‐
Referenceitem(µg):	2‐amino‐anthracene	2.5	1633	1461	1590	1561.3	89.5	13.5
		5.000	86	79	78	81.0	4.4	0.7
		1.667	89	107	94	96.7	9.3	0.8
TestitemµL		0.556	102	101	96	99.7	3.2	0.9
		0.185	93	85	92	90.0	4.4	0.8
		0.062	87	118	110	105.0	16.1	0.9

 

TA1535

Solvent:	Cornoil	‐	17	29	23	23.0	6.0	‐
Referenceitem(µg):	2‐amino‐anthracene	30	413	365	383	387.0	24.2	16.8
		5.000	10	26	13	16.3	8.5	0.7
		1.667	16	14	15	15.0	1.0	0.7
TestitemµL		0.556	21	23	19	21.0	2.0	0.9
		0.185	24	15	30	23.0	7.5	1.0
		0.062	20	17	18	18.3	1.5	0.8

 

TA1537

Solvent:	Cornoil	‐	7	7	11	8.3	2.3	‐
Referenceitem(µg):	2‐amino‐anthracene	2.5	221	231	222	224.7	5.5	27.0
		5.000	5	5	4	4.7	0.6	0.6
		1.667	8	5	3	5.3	2.5	0.6
TestitemµL		0.556	5	11	6	7.3	3.2	0.9
		0.185	9	8	4	7.0	2.6	0.8
		0.062	13	4	8	8.3	4.5	1.0

 

WP2

Solvent:	Cornoil	‐	283	278	232	264.3	28.1	‐
Referenceitem(µg):	2‐amino‐anthracene	30	1979	1893	1746	1872.7	117.8	7.1
		5.000	316	341	227	294.7	59.9	1.1
		1.667	347	240	237	274.7	62.7	1.0
TestitemµL		0.556	329	329	240	299.3	51.4	1.1
		0.185	313	318	274	301.7	24.1	1.1
		0.062	264	262	234	253.3	16.8	1.0

 

 

Applicant's summary and conclusion

Conclusions:

The test item CAPRATE D’ETHYLE does not induce point mutations or frameshifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure over the concentration range tested. Therefore, the test item CAPRATE D’ETHYLE at an exposure dose range of 0,062 – 5,0 μL/plate is considered to be NON MUTAGENIC / NON PRO- MUTAGENIC under the experimental conditions assayed.

Executive summary:

The GLP compliant test was performed in accordance with OECD Guideline 471 for the Testing of Chemicals (Bacterial Reverse Mutation Test. Adopted 21st July 1997) in order to assess the potential mutagenicity toxicity on bacteria.

Cytotoxicity evaluation of the test item was performed in the S. typhimurium TA100 strain by the direct incorporation procedure and without metabolic activation with 5 concentrations of the test item based on its solubility profile (ranging from 0,062 to 5,0 μL/plate). No test item related cytotoxic activity was observed at any of the concentrations tested.

On the basis of these results, 5 test item doses ranging from 0,062 to 5,0 μL/plate were assayed in the main test, corn oil mixing with PBS was used as vehicle. Direct incorporation and preincubation method were used, with and without metabolic activation system (S9 fraction, from rats liver induced with intravenous administration of Aroclor 1254). Plates were incubated with test item 48 hours and reverant colonies were counted.

Overall interpretation of the study results suggests that the test item does not induce point mutations or frameshifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure.

Therefore, the test item CAPRATE D’ETHYLE at an exposure dose range of 0,062 – 5,0 μL/plate is considered to be NON MUTAGENIC / NON PRO- MUTAGENIC under the experimental conditions assayed.