Ethyl 2-cyclohexylpropionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1 to 28 November 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD Guideline 471 and it is GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta-S.typhimurium: The Salmonella typhimurium reverse mutation assay.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-cyclohexylpropionate

EC Number:

412-280-5

EC Name:

Ethyl 2-cyclohexylpropionate

Cas Number:

2511-00-4

Molecular formula:

C11H20O2

IUPAC Name:

ethyl 2-cyclohexylpropanoate

Details on test material:

- Physical state: clear, colourless liquid
- Storage condition of test material: 4ºC in the dark

Method

Target gene:

S. typhimurium TA 1535 hisG46rfa uvrB
S. typhimurium TA 1537 hisC3076 rfa uvrB
S. typhimurium TA 1538 hisD3052 rfa uvrB
S. typhimurium TA 98 hisD3052 rfa uvrB pKM101
S. typhimurium TA 100 hisG46 rfa uvrB pKM101

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was prepared by administration of Aroclor 1254 in a single intra-peritonal injection (in Arachis oil, 500 mg/kg bw)

Test concentrations with justification for top dose:

In the preliminary toxicity study, four concentrations of test subtance were assessed for toxicity using the five tester strains. The highest concentration was 50 mg/ml of test subtance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Three 10-fold serial dilutions of the highest concentration were also tested. The chosen solvent is ethanol.
In the mean mutation study, the test substance was added to culture of the five tester strains at five concentrations separated by c.half-log10 intervals. The highest concentration of the substance used was 150µg/plate. The positive control compounds were also included.

Vehicle / solvent:

ethanol

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(5µg/plate) TA1535 and (3µg/plate) TA100 without S9mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(80µg/plate) TA1537 without S9mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

(2µg/plate) TA1538 and (1µg/plate) TA98 without S9mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(2µg/plate) TA1535 and TA 1537 and (0.5µg/plate) TA1538 and TA98 and (1µg/plate) strain TA100 with S9mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: two independent experiments were performed

NUMBER OF PLATES EVALUATED: three per dose

DETERMINATION OF CYTOTOXICITY
- Method: any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

The mean number of relevant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test material is assessed by applying the following criteria.
a) If treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
b) If treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the material's mutagenic in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrow dose range than that already tested; the use of diferrent levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the material is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989).

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the first mutation test toxicity was observed at 150 µg/plate towards all the tester strains except TA1535

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the first mutation test toxicity was observed at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The revertant colony counts for the substance obtained in the preliminary toxicity test are shown in Table 1. The substance was toxic towards the tester strains at both 5000 µg/plate. Therefore 150 µg/plate was chosen as the top dose level in the mutation tests.
The mean number of revertant colonies, together with the individual plate counts for the substance obtained in the first mutation test with the tester strains are shown in Table 2. Positive control mutability checks are shown in Table 3.
In the first mutation test toxicity was observed at 150 µg/plate towards all the tester strains except TA 1535.
The mean number of revertant colonies, together with the individual plate counts for the substance obtained in the second mutation test with the tester strains are shown in Table 4. Positive control mutability checks are shown in Table 5.
No substantial increases in revertant colony number of any of the tester strains were observed following treatment with the substance at any dose level, either in the presence of S-9 mix.
The concurrent positive control compound demostrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Dose range finding test on the test substance - revertant colony counts obtaned with bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA100.
Dose Level (µg/plate)	Metabolic activation	Bacterial Strains
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
5000	-	IL	IL	IL	IL	IL
500	-	IL	IL	IL	IL	IL
50	-	6	13	9	21	83
5	-	11	12	14	25	110
Solvent	-	17	18	12	25	110
5000	+	IL	IL	IL	IL	IL
500	+	IL	IL	IL	IL	IL
50	+	12	15	16	23	104
5	+	14	14	9	24	119
Solvent	+	16	15	19	24	107
Table 2: Mutation Test 1
Test substance - revertant colony counts obtained per plate using bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.
Strain	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts	SD	Individual revertant colony counts
TA 1535	150.0	-	9	2.1	7	11	10
	50.0	-	12	1.5	13	10	12
	15.0	-	11	1.2	12	12	10
	5.0	-	12	4.2	13	15	7
	1.5	-	10	2.6	7	11	12
	0.0	-	12	2.0	14	10	12
	Solvent	-	14	0.6	14	13	14
	150.0	+	6	1.7	7	4	7
	50.0	+	14	1.5	14	13	16
	15.0	+	15	2.3	14	14	18
	5.0	+	12	1.5	11	14	12
	1.5	+	15	1.5	16	15	13
	0.0	+	14	5.0	19	14	9
	Solvent	+	16	2.5	13	18	16
TA 1537	150.0	-	-	-	IL	IL	IL
	50.0	-	13	1.5	13	15	12
	15.0	-	15	2.6	17	16	12
	5.0	-	10	3.2	14	8	9
	1.5	-	12	4.6	17	9	9
	0.0	-	17	1.2	18	16	18
	Solvent	-	15	1.5	17	15	14
	150.0	+	-	-	IL	IL	IL
	50.0	+	18	-	c	20	16
	15.0	+	14	4.7	9	16	18
	5.0	+	13	1.0	14	12	13
	1.5	+	17	2.6	16	20	15
	0.0	+	18	2.1	16	17	20
	Solvent	+	17	2.5	15	20	17
TA 1538	150.0	-	-	-	IL	IL	IL
	50.0	-	10	2.0	8	12	10
	15.0	-	11	3.5	7	13	13
	5.0	-	11	3.6	7	14	12
	1.5	-	14	2.6	12	13	17
	0.0	-	15	3.1	14	18	12
	Solvent	-	12	2.6	14	13	9
	150.0	+	-	-	IL	IL	IL
	50.0	+	14	0.6	13	14	14
	15.0	+	13	1.0	12	14	13
	5.0	+	15	2.3	16	16	12
	1.5	+	14	3.2	16	10	15
	0.0	+	16	1.0	16	17	15
	Solvent	+	17	2.6	20	15	16
TA 98	150.0	-	-	-	IL	IL	IL
	50.0	-	20	1.5	20	21	18
	15.0	-	24	4.6	29	21	21
	5.0	-	24	3.5	24	21	28
	1.5	-	24	2.3	25	25	21
	0.0	-	22	2.1	24	23	20
	Solvent	-	25	1.5	25	24	27
	150.0	+	-	-	IL	IL	IL
	50.0	+	24	2.1	22	25	26
	15.0	+	22	2.1	20	24	23
	5.0	+	26	3.5	22	28	28
	1.5	+	26	4.6	21	27	30
	0.0	+	24	4.7	28	26	19
	Solvent	+	29	3.5	26	33	29
TA 100	150.0	-	-	-	IL	IL	IL
	50.0	-	110	6.5	116	110	103
	15.0	-	107	0.0	107	107	107
	5.0	-	99	10.1	104	105	87
	1.5	-	110	8.7	120	105	105
	0.0	-	99	9.0	90	99	108
	Solvent	-	101	14.0	85	107	111
	150.0	+	-	-	IL	IL	IL
	50.0	+	118	2.5	115	118	120
	15.0	+	118	6.6	112	117	125
	5.0	+	106	6.7	112	99	108
	1.5	+	115	6.2	110	113	122
	0.0	+	130	2.5	127	132	130
	Solvent	+	117	7.5	117	110	125
Table 3: Mutation Test 1
Mutability tests with bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Strain	Compound	Dose Level (µg/plate)	Metabolic activation	Mean revertant colony counts	SD	Individual revertant colony counts
TA 1535	EENG	5.0	-	211	24.8	226	182	224
TA 1537	9 AC	80.0	-	-	-	X	X	X
TA 1538	NF	2.0	-	71	2.6	68	72	73
TA 98	NF	1.0	-	116	20.6	96	137	114
TA 100	ENNG	3.0	-	308	11.6	314	295	316
TA 1535	AA	2.0	+	215	16.0	232	214	200
TA 1537	AA	2.0	+	111	13.0	112	124	98
TA 1538	AA	0.5	+	134	22.2	108	147	146
TA 98	AA	0.5	+	198	25.5	224	173	196
TA 100	AA	1.0	+	422	16.1	407	420	439
Table 4: Mutation Test 2
Test substance - revertant colony counts obtained per plate using bacterial strains TA1535, TA1537, TA1538, TA98 and TA100
Strain	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts	SD	Individual revertant colony counts
TA 1535	150.0	-	13	4.0	17	9	12
	50.0	-	11	0.6	11	11	12
	15.0	-	10	1.0	9	10	11
	5.0	-	11	3.6	15	8	10
	1.5	-	12	2.0	10	14	12
	0.0	-	11	2.5	11	9	14
	Solvent	-	12	2.9	14	14	9
	150.0	+	17	3.1	16	14	20
	50.0	+	13	2.6	12	16	11
	15.0	+	14	1.2	15	13	15
	5.0	+	17	1.5	19	16	17
	1.5	+	14	3.6	13	18	11
	0.0	+	14	2.1	12	15	16
	Solvent	+	13	0.0	13	13	13
TA 1537	150.0	-	12	2.5	12	14	9
	50.0	-	12	3.2	13	8	14
	15.0	-	14	3.1	13	11	17
	5.0	-	14	3.8	12	11	18
	1.5	-	14	1.5	13	16	14
	0.0	-	10	4.5	6	10	15
	Solvent	-	15	1.2	16	16	14
	150.0	+	14	3.8	18	12	11
	50.0	+	12	2.9	10	10	15
	15.0	+	14	0.6	14	15	14
	5.0	+	11	0.6	12	11	11
	1.5	+	15	1.5	13	16	15
	0.0	+	15	1.7	13	16	16
	Solvent	+	15	0.0	15	15	15
TA 1538	150.0	-	9	4.4	14	6	7
	50.0	-	7	5.0	7	2	12
	15.0	-	7	1.2	6	6	8
	5.0	-	8	1.5	8	10	7
	1.5	-	11	1.0	12	11	10
	0.0	-	9	4.0	5	13	10
	Solvent	-	12	2.3	11	11	15
	150.0	+	18	1.5	19	16	18
	50.0	+	14	0.6	14	15	14
	15.0	+	17	2.6	20	15	16
	5.0	+	16	2.5	14	19	16
	1.5	+	16	2.5	18	16	13
	0.0	+	17	2.6	19	18	14
	Solvent	+	15	2.5	18	13	15
TA 98	150.0	-	21	0.6	22	21	21
	50.0	-	20	1.5	19	20	22
	15.0	-	26	5.2	23	23	32
	5.0	-	21	2.1	23	20	19
	1.5	-	25	8.5	17	25	34
	0.0	-	23	5.5	20	29	19
	Solvent	-	26	3.8	23	30	24
	150.0	+	27	2.9	24	29	29
	50.0	+	29	4.5	29	34	25
	15.0	+	32	2.5	29	34	32
	5.0	+	31	1.0	30	32	31
	1.5	+	25	1.7	26	23	26
	0.0	+	34	-	c	31	36
	Solvent	+	22	2.3	21	25	21
TA 100	150.0	-	78	4.7	83	74	76
	50.0	-	87	9.7	95	89	76
	15.0	-	88	6.7	80	91	92
	5.0	-	81	5.1	87	77	80
	1.5	-	86	3.2	84	85	90
	0.0	-	98	17.6	78	108	109
	Solvent	-	94	10.2	106	90	84
	150.0	+	103	6.9	111	99	99
	50.0	+	108	11.9	104	98	121
	15.0	+	99	2.5	101	99	96
	5.0	+	105	8.6	103	97	114
	1.5	+	111	4.0	115	107	111
	0.0	+	109	2.6	111	110	106
	Solvent	+	105	5.3	99	107	109
Table 5: Mutation test 2
Mutability tests with bacterial strains TA1535, TA1537, TA1538, TA98 and TA100
Strain	Compound	Dose Level (µg/plate)	Metabolic activation	Mean revertant colony counts	SD	Individual revertant colony counts
TA 1535	EENG	5.0	-	336	35.8	367	297	345
TA 1537	9 AC	80.0	-	-	-	x	x	x
TA 1538	NF	2.0	-	62	12.1	75	51	61
TA 98*	NF	1.0	-	82	4.2	85	83	77
TA 100	ENNG	3.0	-	294	25.2	323	278	281
TA 1535	AA	2.0	+	281	19.6	259	286	297
TA 1537	AA	2.0	+	93	15.7	96	107	76
TA 1538	AA	0.5	+	219	26.1	246	217	194
TA 98*	AA	0.5	+	135	11.8	138	122	145
TA 100	AA	1.0	+	807	56.5	790	761	870
- Absence	C Contaminated
+ Presence	SD Standard deviation
IL Incomplete bacterial lawn
x Too many colonies to count accurately
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
9 AC 9-aminoacridine
NF 2-nitrofluorene
AA 2-aminoanthracene
* Data obtained from a separate experiment due to contamination

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that, when tested in ethanol, the substance shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of the substance, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA100) were exposed to the test material, diluted in ethanol which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary dose range finding study with dose levels of up to 5000 µg/plate toxicity was observed towards the tester strains at both 5000 and 500 µg/plate. A top dose level of 150 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 50, 15, 5, 1.5 µg/plate.

No evidence of mutagenic activity was seen at any dose level of the substance in either mutation test.

The concurrent positive control compounds demostrated the sensivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in ethanol, the substance was not mutagenic in this bacterial system.