4-propylcyclohexanone

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:

1990-05-16 to 1990-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-propylcyclohexanone

EC Number:

406-810-4

EC Name:

4-propylcyclohexanone

Cas Number:

40649-36-3

Molecular formula:

C9H16O

IUPAC Name:

4-propylcyclohexan-1-one

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA 98, TA 1538, TA 1537

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Male Chbbr:Thom (Wistar) rats (aged 6-8 weeks) are given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) diluted in Miglyol 812 oil.
- method of preparation of S9 mix: The livers are homogenized in a sterile glass potter homogenizer containing 3 mL of 0.15 M KCl per each gram of liver wet-weight. The homogenate is spun at 9000 x g for 15 minutes at about + 4 °C and the supernatant fluid is decanted and transferred into sterilized and precooled plastic tubes. The S-9 is then frozen in liquid nitrogen and stored at -196 °C.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL

Test concentrations with justification for top dose:

First series of experiments: 50, 250, 1250, 2500, 5000, and 7500 µg/plate.
Second series of experiments: 10, 50, 250, 1250, 2500, and 5000 µg/plate.
The test material concentrations used were selected according to the EEC Guideline and the OECD Guideline for this test system. They were chosen to cover a broad concentration range including, if possible, also a toxic concentration.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin (DAUN); -S-9 mix: TA 98

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1-ethyl-2-nitro-3-nitrosoguanldine (ENNG); -S-9 mix: TA 100, TA 1535

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine (4-NP); -S-9 mix: TA 1538

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 8

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA); +S-9 mix: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 8 plates (4 with and 4 without S-9) were used for each concentration step of the test material and 2-AA, 4 plates, each, for the other control compounds, 16 plates (8 with and 8 without S-9) were used for the negative controls (solvent).
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants, clearing of the background lawn, reduced survival of treated cultures

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:

cytotoxicity

Remarks:

at 5000 µg/plate (-S9/+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (-S9/+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate (-S9/+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Remarks:

at 5000 µg/plate (-S9/+S)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (-S9/+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In a range finding test (using S.typh. TA 100 and TA 1535 without metabolic activation) toxicity was investigated in a wide range of test material concentrations (50 - 10 000 ug/plate). Toxicity normally becomes evident by a reduction in the number of spontaneous revertants, a clearing of the background lawn, or by reduced survival of treated cultures. These effects were observed with Licristal BICH 3 Stage 3 in the range finding test at > 5000 ug/plate. Therefore in the first series of experiments, 6 different amounts of the test material 50, 250, 1250, 2500, 5000, and 7500 ug/plate were used. As for the highest concentration tested cytotoxic effects still were observed in all tester strains, the concentration range for the second series of experiments was changed to 10, 50, 250, 1250, 2500, and 5000 ug/plate.

Any other information on results incl. tables

Table 1: Result of the first series (TA 98)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	17	42	21	17	4	24	12.0	-	-
	50	28	41	32	32	4	33	5.5	-	-
	250	19	29	19	20	4	22	4.9	-	-
	1250	27	26	21	21	4	24	3.2	-	-
	2500	11	17	16	17	4	15	2.9	-	-
	5000	4	11	2	3	4	5	4.1	-	+
	7500	0	0	0	0	4	0	0.0	-	+
	0	30	18	21	42	4	28	10.8	-	-
2-AA	1.0	31	39	33	28	4	33	4.6	-	-
Ve-H2O		28	34	32	27	4	30	3.3	-	-
DAUN	2.0	512	526	530	542	4	528	12.4	-	-
2-NF	2.0	229	192	183	222	4	207	22.4	-	-
	With S-9 Mix
	0	28	35	26	27	4	29	4.1	-	-
	50	20	21	41	31	4	28	9.8	-	-
	250	39	41	42	30	4	38	5.5	-	-
	1250	34	22	32	28	4	29	5.3	-	-
	2500	18	26	27	33	4	26	6.2	-	-
	5000	11	11	7	3	4	8	3.8	-	+
	7500	0	0	0	0	4	0	0.0	-	+
	0	35	34	30	32	4	33	2.2	-	-
2-AA	1.0	703	636	795	734	4	717	66.2	-	-

Table 2: Results of the first series (TA 100)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	168	153	140	124	4	146	18.7	-	-
	50	136	135	138	167	4	144	15.4	-	-
	250	143	165	137	124	4	142	17.1	-	-
	1250	123	141	126	155	4	136	14.8	-	-
	2500	104	99	122	96	4	105	11.6	-	-
	5000	78	84	47	79	4	72	16.9	-	±
	7500	68	71	47	56	4	61	11.1	-	+
	0	154	126	157	126	4	141	17.1	-	-
2-AA	1.0	139	141	140	139	4	140	1.0	-	-
MSS	500.0	930	1132	1294	1112	4	1117	148.9	-	-
ENNG	4.0	653	631	625	595	4	626	23.9	-	-
	With S-9 Mix
	0	194	166	199	167	4	182	17.4	-	-
	50	188	169	167	153	4	169	14.4	-	-
	250	181	182	178	166	4	177	7.4	-	-
	1250	142	139	151	136	4	142	6.5	-	-
	2500	136	155	143	139	4	143	8.3	-	-
	5000	81	113	86	93	4	93	14.1	-	±
	7500	0	0	0	0	4	0	0.0	-	+
	0	163	155	161	156	4	159	3.9	-	-
2-AA	1.0	880	1043	1174	1044	4	1035	120.4	-	-

Table 3: Results of the first series (TA 1535)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	13	3	10	9	4	9	4.2	-	-
	50	7	5	5	9	4	7	1.9	-	-
	250	7	7	10	7	4	8	1.5	-	-
	1250	12	7	6	10	4	9	2.8	-	-
	2500	2	5	8	6	4	5	2.5	-	-
	5000	2	2	2	2	4	2	0.0	-	±
	7500	0	0	0	0	4	0	0.0	-	±
	0	8	10	5	4	4	7	2.8	-	-
2-AA	1.0	8	8	9	7	4	8	0.8	-	-
ENNG	10.0	503	436	496	568	4	501	54.0	-	-
Ve-H2O		10	4	9	6	4	7	2.8	-	-
NaN3	1.0	375	378	370	352	4	369	11.6	-	-
	With S-9 Mix
	0	5	10	8	12	4	9	3.0	-	-
	50	9	4	13	11	4	9	3.9	-	-
	250	5	8	11	8	4	8	2.4	-	-
	1250	7	7	8	5	4	7	1.3	-	-
	2500	9	9	3	3	4	6	3.5	-	-
	5000	1	1	0	2	4	1	0.8	-	+
	7500	0	0	0	0	4	0	0.0	-	+
	0	14	11	8	7	4	10	3.2	-	-
2-AA	1.0	117	140	83	162	4	126	33.8	-	-

Table 4: Results of the first series (TA 1537)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	3	4	3	8	4	5	2.4	-	-
	50	6	3	5	3	4	4	1.5	-	-
	250	5	2	5	5	4	4	1.5	-	-
	1250	4	2	3	3	4	3	0.8	-	-
	2500	2	1	1	5	4	2	1.9	-	-
	5000	0	0	0	0	4	0	0.0	-	±
	7500	0	0	0	0	4	0	0.0	-	+
	0	6	4	5	6	4	5	1.0	-	-
2-AA	1.0	3	6	5	6	4	5	1.4	-	-
EtOH		2	6	3	2	4	3	1.9	-	-
9-AA	20.0	23	33	37	31	4	31	5.9	-	-
9-AA	50.0	117	129	109	157	4	128	21.0	-	-
	With S-9 Mix
	0	4	8	5	6	4	6	1.7	-	-
	50	10	6	6	4	4	7	2.5	-	-
	250	4	7	4	6	4	5	1.5	-	-
	1250	4	5	6	5	4	5	0.8	-	-
	2500	5	2	4	4	4	4	1.3	-	-
	5000	2	2	0	2	4	2	1.0	-	±
	7500	0	0	0	0	4	0	0.0	-	+
	0	7	9	5	5	4	7	1.9	-	-
2-AA	1.0	57	57	74	61	4	62	8.1	-	-

Table 5: Results of the first series (TA 1538)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	5	4	6	4	4	5	1.0	-	-
	50	5	5	7	6	4	6	1.0	-	-
	250	5	3	10	7	4	6	3.0	-	-
	1250	5	3	4	10	4	6	3.1	-	-
	2500	4	4	4	6	4	5	1.0	-	-
	5000	3	1	0	2	4	2	1.3	-	±
	7500	0	0	0	0	4	0	0.0	-	+
	0	5	7	6	4	4	6	1.3	-	-
2-AA	1.0	11	7	9	8	4	9	1.7	-	-
4-NP	10.0	492	336	392	412	4	408	64.6	-	-
2-NF	5.0	599	611	677	624	4	628	34.4	-	-
	With S-9 Mix
	0	5	7	10	11	4	8	2.8	-	-
	50	8	9	7	9	4	8	1.0	-	-
	250	6	6	8	8	4	7	1.2	-	-
	1250	5	11	7	5	4	7	2.8	-	-
	2500	5	4	6	7	4	6	1.3	-	-
	5000	0	4	2	0	4	2	1.9	-	±
	7500	0	0	0	0	4	0	0.0	-	+
	0	5	8	7	14	4	9	3.9	-	-
2-AA	1.0	425	429	492	459	4	451	31.1	-	-

Table 6: Results of the second series (TA 98)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	18	33	27	29	4	27	6.3	-	-
	10	18	29	26	27	4	25	4.8	-	-
	50	22	41	28	26	4	29	8.2	-	-
	250	16	33	20	16	4	21	8.1	-	-
	1250	17	33	28	21	4	25	7.1	-	-
	2500	19	17	14	21	4	18	3.0	-	-
	5000	11	13	9	7	4	10	2.6	-	+
	0	29	39	22	27	4	29	7.1	-	-
2-AA	1.0	32	28	34	31	4	31	2.5	-	-
Ve-H2O		28	27	18	18		23	5.5	-	-
DAUN	2.0	1000	1063	1189	1045	4	1074	81.0	-	-
2-NF	2.0	253	254	259	272	4	260	8.7	-	-
	With S-9 Mix
	0	31	29	32	33	4	31	1.7	-	-
	10	42	32	42	34	4	38	5.3	-	-
	50	32	26	28	30	4	29	2.6	-	-
	250	48	32	28	35	4	36	8.7	-	-
	1250	35	31	42	26	4	34	6.8	-	-
	2500	19	35	30	22	4	27	7.3	-	-
	5000	7	3	5	6	4	5	1.7	-	+
	0	46	43	31	34	4	39	7.1	-	-
2-AA	1.0	425	428	409	414	4	419	9.0	-	-

Table 7: Results of the second series (TA 100)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	152	176	154	161	4	161	10.9	-	-
	10	154	157	152	190	4	163	18.0	-	-
	50	179	137	150	136	4	151	20.0	-	-
	250	126	183	174	176	4	165	26.1	-	-
	1250	157	148	125	126	4	139	16.0	-	-
	2500	152	105	125	148	4	133	21.9	-	-
	5000	111	99	109	123	4	111	9.8	-	±
	0	144	152	175	167	4	160	14.1	-	-
2-AA	1.0	164	167	156	175	4	166	7.9	-	-
MMS	500.0	1152	1417	1190	1177		1234	123.0	-	-
ENNG	4.0	935	836	986	962	4	930	65.9	-	-
	With S-9 Mix
	0	169	178	183	149	4	170	15.0	-	-
	10	182	175	182	182	4	180	3.5	-	-
	50	192	176	168	206	4	186	16.9	-	-
	250	153	187	183	190	4	178	17.1	-	-
	1250	194	174	187	178	4	183	9.0	-	-
	2500	180	165	154	181	4	170	12.9	-	-
	5000	107	97	96	100	4	100	5.0	-	±
	0	195	189	151	194	4	182	21.0	-	-
2-AA	1.0	675	592	661	610	4	635	39.8	-	-

Table 8: Results of the second series (TA 1535)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	18	8	13	13	4	13	4.1	-	-
	10	15	15	17	18	4	16	1.5	-	-
	50	8	15	17	17	4	14	4.3	-	-
	250	18	20	17	15	4	18	2.1	-	-
	1250	14	19	18	18	4	17	2.2	-	-
	2500	8	8	16	13	4	11	3.9	-	-
	5000	9	8	9	5	4	8	1.9	-	±
	0	17	20	16	16	4	17	1.9	-	-
2-AA	1.0	15	17	17	20	4	17	2.1	-	-
ENNG	10.0	639	646	541	543	4	592	58.1	-	-
Ve-H2O		19	7	c	18	4	15	6.7	-	-
NaN3	1.0	451	364	414	391	4	405	36.9	-	-
	With S-9 Mix
	0	17	27	21	21	4	22	4.1	-	-
	10	15	16	20	15	4	17	2.4	-	-
	50	21	16	22	28	4	22	4.9	-	-
	250	9	27	20	17	4	18	7.5	-	-
	1250	30	26	19	15	4	23	6.8	-	-
	2500	8	7	7	9	4	8	1.0	-	-
	5000	5	4	3	3	4	4	1.0	-	+
	0	19	26	30	20	4	24	5.2	-	-
2-AA	1.0	118	138	99	113	4	117	16.1	-	-

Table 9: Results of the second series (TA 1537)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	8	5	6	9	4	7	1.8	-	-
	10	9	4	5	5	4	6	2.2	-	-
	50	6	9	6	8	4	7	1.5	-	-
	250	6	7	3	4	4	5	1.8	-	-
	1250	5	3	7	3	4	5	1.9	-	-
	2500	5	2	1	4	4	3	1.8	-	-
	5000	1	0	0	1	4	1	0.6	-	±
	0	5	7	4	4	4	5	1.4	-	-
2-AA	1.0	8	8	5	5	4	7	1.7	-	-
EtOH		3	5	7	7	4	6	1.9	-	-
9-AA	20.0	28	29	23	30	4	28	3.1	-	-
9-AA	50.0	149	169	144	149	4	153	11.1	-	-
	With S-9 Mix
	0	6	6	7	5	4	6	0.8	-	-
	10	7	6	7	9	4	7	1.3	-	-
	50	4	8	5	8	4	6	2.1	-	-
	250	8	10	8	5	4	8	2.1	-	-
	1250	5	2	5	5	4	4	1.5	-	-
	2500	4	4	2	2	4	3	1.2	-	-
	5000	3	0	0	1	4	1	1.4	-	+
	0	6	9	6	4	4	6	2.1	-	-
2-AA	1.0	73	59	67	63	4	66	6.0	-	-

Table 10: Results of the second series (TA 1538)

Conc. (µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
Without S-9 Mix
	0	6	8	7	7	4	7	0.8	-	-
	10	5	6	8	5	4	6	1.4	-	-
	50	5	7	10	6	4	7	2.2	-	-
	250	5	9	10	4	4	7	2.9	-	-
	1250	10	8	8	5	4	8	2.1	-	-
	2500	3	4	3	3	4	3	0.5	-	-
	5000	0	3	1	1	4	1	1.3	-	±
	0	7	12	9	9	4	9	2.1	-	-
2-AA	1.0	9	6	5	9	4	7	2.1	-	-
4-NP	10.0	774	717	1064	653	4	802	181.5	-	-
2-NF	5.0	709	1165	977	903	4	939	188.6	-	-
	With S-9 Mix
	0	12	7	8	14	4	10	3.3	-	-
	10	16	13	16	8	4	13	3.8	-	-
	50	9	14	9	7	4	10	3.0	-	-
	250	8	8	4	6	4	7	1.9	-	-
	1250	7	5	3	4	4	5	1.7	-	-
	2500	3	4	6	4	4	4	1.3	-	-
	5000	2	0	0	1	4	1	1.0	-	+
	0	9	10	11	8	4	10	1.3	-	-
2-AA	1.0	405	416	343	357	4	380	35.7	-	-

Bg = reduction of background growth (toxicity, multiple pinpoint colonies)

Pc = precipitation of the test material in the top agar layer

c = contaminated

- = not observed

+ = strong effect

± = weak effect

2-AA = 2-aminoanthracene
9-AA = 9-aminoacridine
DAUN = daunomycin
DMSO = dimethylsulfoxide
ENNG = 1-ethyl-2-nitro-3-nitrosoguanidine
EtOH = ethanol
MMS = methyl methanesulfonate
NaN3 = sodium azide
2-NF = 2-nitrofluorene
4-NP = 4-nitro-1,2-phenylene diamine
Ve-H20 = deionized water

Applicant's summary and conclusion

Conclusions:

Under the test conditions used, the test item was not mutagenic in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 with or without addition of S9 mix.

Executive summary:

The investigations for mutagenic potential were performed according OECD TG 471 using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, and TA 1538 as tester strains. The plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used.
The substance was tested in two series of experiments at the following concentrations:
First series of experiments: 50, 250, 1250, 2500, 5000, and 7500 µg/plate.
Second series of experiments: 10, 50, 250, 1250, 2500, and 5000 µg/plate.
9-Aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanldine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, and sodium azide served as positive control compounds for testing the bacteria. 2-Aminoanthracene was used for testing the bacteria and the activity of the S-9 preparation.
The positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used. With and without addition of S-9 as the metabolizing system, the test substance did not show any mutagenic activity in the concentration range used.