4-trans-Pentyl-[1,1-bicyclohexyl]-4-on

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:

07-05-1991 to 03-06-1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1's,4'r)-4'-pentyl-[1,1'-bi(cyclohexane)]-4-one

EC Number:

617-638-6

Cas Number:

84868-02-0

Molecular formula:

C17H30O

IUPAC Name:

(1's,4'r)-4'-pentyl-[1,1'-bi(cyclohexane)]-4-one

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

TA 100 (his G 46, uvrB, rfa + R-factor)
TA 1535 (his G 46, uvrB, rfa)
TA 98 (his D 3052, uvrB, rfa + R-factor)
TA 1537 (his C 3076, uvrB, rfa)

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

TA 1538 (his D 3052, uvrB, rfa)

Metabolic activation:

with and without

Metabolic activation system:

S-9 metabolizing system (S-9 mix)
Preparation of liver homogenate fraction (S-9)
S-9 is routinely prepared, following the proposals of AMES et al. (1975). It is kept at about -196 °C. Male Chbb: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. The animals receive drinking water and Altromin standard diet ad libitum. About 16 hours before sacrifice, the feed is removed. On day 5 the rats are sacrificed and the livers collected in ice-cooled sterilized beakers containing 0.15 M KCl. 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 9000xg 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. The protein content of the S-9 preparation is determined by the biuret method.

Test concentrations with justification for top dose:

Concentrations in the range-finding test: 50, 250, 500, 1000, 2000, 3000, 4000, 5000, 7500, 10000 µg/plate
Concentrations in Experiment 1: 50, 250, 1250, 2500, 5000, 10000 µg/plate
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 - 10000 µg/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 not observed with the test item in the range-finding test. Precipitation of the test material did not interfere with the bacterial growth. Therefore, 6 different amounts of the test material up to 10000 µg/plate were used in each of the two series of experiments.

Vehicle / solvent:

- Solvent: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene diamine 4-NP, daunomycin DAUN, 1-ethyl-2-nitro-3-nitrosoguanidine ENNG

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 8
- 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
- Incubation temperature: 37 °C

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:

no cytotoxicity

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The results of both series of experiments, each performed with and without the addition of rat liver microsomal fraction as external metabolizing system, were negative. 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. The negative control mutant frequencies were all in the regular range, and the positive control compounds yielded the expected mutant frequencies that were greatly in excess of the background.

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of four plates), Experiment 1

	strain TA 100		strain TA 1535		strain TA 1537		strain TA 1538		strain TA 98
conc. [µg] per plate	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	148	163	10	18	6	8	8	12	21	30
50	138	191	9	13	6	6	6	11	21	31
250	144	176	9	13	5	5	7	12	15	28
1250	139	151	9	14	6	8	6	8	18	25
2500	154	167	8	15	5	5	6	9	17	27
5000	153	164	8	12	4	6	8	9	18	29
10000	139	163	11	11	5	5	6	8	17	27
0	135	174	11	16	7	8	8	12	25	26
NaN3 (1 µg/pl)			622
DAUN (2 µg/pl)									497
2-NF (2 µg/pl)									301
2-NF (5 µg/pl)							5
2-AA (1 µg/pl)	157	576	8	139	5	60	7	454	19	452
Ve-H2O			15						20
MMS (500 µg/pl)	1157
ENNG (4 µg/pl)	929		423
Etanol EtOH					6
9-AA (20 µg/pl)					30
9-AA (50 µg/pl)					283
4-NP (10 µg/pl)							1026

NaN3: Sodium azide, DAUN: Daunomycin, 2-NF: 2-Nitrofluorene, 2-AA: 2-Aminoanthracene, Ve-H2O: deionized water, MMS: methylmethanesulfonate, ENNG: 1-ethyl-2-nitro-3-nitrosoguanidine, 9-AA: 9-Aminoacridine, 4-NP: 4-nitro-1,2-phenylene diamine

 

Table 2: Number of revertants per plate (mean of four plates), Experiment 2

	strain TA 100		strain TA 1535		Strain TA 1537		strain TA 1538		strain TA 98
conc. [µg]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	156	178	8	15	5	5	6	12	17	18
50	145	170	8	11	5	5	4	7	13	25
250	144	181	6	13	4	5	6	9	13	25
1250	161	173	6	12	4	7	5	8	15	20
2500	142	165	7	11	4	4	6	10	16	23
5000	145	164	7	11	3	4	4	8	14	19
10000	140	167	5	10	4	3	3	9	14	19
0	156	167	9	13	5	6	6	14	15	28
NaN3 (1 µg/pl)			388
DAUN (2 µg/pl)									659
2-NF (2 µg/pl)									301
2-NF (5 µg/pl)							744
2-AA (1 µg/pl)	148	640	8	136	4	53	5	429	16	381
Ve-H2O			8						20
MMS (500 µg/pl)	966
ENNG (4 µg/pl)	880		554
Etanol EtOH					5
9-AA (20 µg/pl)					22
9-AA (50 µg/pl)					267
4-NP (10 µg/pl)							855

NaN3: Sodium azide, DAUN: Daunomycin, 2-NF: 2-Nitrofluorene, 2-AA: 2-Aminoanthracene, Ve-H2O: deionized water, MMS: methylmethanesulfonate, ENNG: 1-ethyl-2-nitro-3-nitrosoguanidine, 9-AA: 9-Aminoacridine, 4-NP: 4-nitro-1,2-phenylene diamine 

Applicant's summary and conclusion

Conclusions:

During the experiments there was no evidence of toxicity to the bacteria caused by the test material exposure. The test material is considered to be non-mutagenic.

Executive summary:

The investigations for mutagenic potential were performed using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, and TA 1538 as tester strains according to OECD 471. The plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used. The test item was tested in two series of experiments at the following concentrations: 50, 250, 1250, 2500, 5000, and 10000 µg/plate. 9-Aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, 2-nitro-fluorene, 4-nitro-l,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. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. During the experiments there was no evidence of toxicity to the bacteria caused by the test material exposure. Under the conditions described, there were no relevant increases in revertant numbers after treatment with the test item observed in both, the absence and presence of S9 mix. In conclusion, the test material was considered to be non-mutagenic under the described experimental conditions.