1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthyl)ethan-1-one

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:

12 October - 22 November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study following OECD guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The standard plate incorporation and the preincubation methods

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthyl)ethan-1-one

EC Number:

216-133-4

EC Name:

1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthyl)ethan-1-one

Cas Number:

1506-02-1

Molecular formula:

C18H26O

IUPAC Name:

1-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethyl-2-naphthyl)ethan-1-one

Details on test material:

AHTN

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 4

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S-9 mix

Test concentrations with justification for top dose:

Dose range 8-5000 µg/plate.

Vehicle / solvent:

Dimethyl Sulfoxide (Merck)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

for TA1535 and TA100

Positive control substance:

sodium azide

Remarks:

SERVA, Lot 30175 - Dosage: 1.0µg/plate

Migrated to IUCLID6: without metabolic activation S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

for TA1537 and TA97

Positive control substance:

other: ICR 191 without metabolic activation S9

Remarks:

SERVA, Control K8 - Dosage: 1.0µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

for TA98

Positive control substance:

2-nitrofluorene

Remarks:

ALDRICH, Lot 3734435 - Dosage: 0.5µg/plate

Migrated to IUCLID6: without metabolic activation S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

for TA102

Positive control substance:

mitomycin C

Remarks:

SIGMA, Lot 32 H0326 - Dosage: 0.4µg/plate

Migrated to IUCLID6: without metabolic activation S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

for E. coli WP2 uvr A

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4-Nitroquinolineoxide (SIGMA, Lot 84F-0572)

Migrated to IUCLID6: without metabolic activation S9

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

for all strains to examine the activity of the S9 mix

Positive control substance:

other: 2-Aminoanthracene - with and without metabolic activation S9

Remarks:

SIGMA, Lot 121 H3475

Details on test system and experimental conditions:

The Salmonella typhimurium strains TA1535, TA1537, TA97, TA98, TA100 and TA102 were obtained from B.N. Ames. E. coli WP2 uvrA was obtained from The National Collection of Industrial and Marine Bacteria Ltd, Aberdeen Scotland.

Evaluation criteria:

There was no generally accepted statistical treatment of Ames test and E.coli reversion system data. In most tests the results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535, TA1537, TA98 and E. coli VP2 uvrA. For strains TA97, TA100 and TA1O2 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of bis+ or trp+ revertant colonies.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The results of the standard plate incorporation test are summarized in table 1 and of the preincubation test in table 2.

The spontaneous mutation rate was within the range observed for historical controls. All positive controls gave acceptable responses.

Toxic effects were not apparent in the standard assay, thus a dose range of 8 to 5000 µg/plate was evaluated to include solutions and suspensions.

AHTN did not cause en increase of the number of revertant colonies in any of the seven tester strains.

Table I Salmonella mutagenicity test (standard assay). Mean values and standard deviations.

Experiment No	01	01	01	01	01	01	02	02	02	02	02	02	02	02
Activation	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Strain	TA1535	TA1535	TA1537	TA1537	WP2uvrA	WP2uvrA	TA97	TA97	TA9B	TA98	TA100	TA100	TA102	TA102
Concentration µg/plate
0	13	9	10	10	24	22	245	245	26	28	196	197	312	298
	±5	±3	±4	±4	±8	±6	±20	±3	±6	± 4	±21	±25	±24	±7
8	13	7	7	11	23	20	258	257	24	32	197	207	324	292
	±2	±3	±1	±4	±5	±1	±20	±11	±4	± 7	±12	±16	±20	±19
40	15	12	9	11	19	26	250	251	27	27	179	208	330	314
	±1	±4	±2	±5	±6	±2	±16	±20	± 4	± 5	±15	± 7	±18	± 9
200	13	10	9	9	19	21	252	273	27	36	194	203	312	346
	±5	±3	±1	±2	±5	±5	±17	±25	±4	±11	±14	±17	±27	±17
1000	16	8	8	9	15	21	240	231	25	28	197	176	307	291
	±3	±2	±1	±2	±2	±6	±19	±30	± 5	± 5	±14	±24	±16	±19
5000	14	6	6	4	21	23	237	234	21	26	203	199	278	319
	±4	±4	±1	±2	±4	±5	±23	±20	± 4	± 2	± 8	± 8	±13	±30

Table 2 Salmonella mutagenicity test (Liquid preincubation assay). Mean values and standard deviations.

Experiment No	01	01	01	01	01	01	02	02	02	02	02	02	02	02
Activation	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Strain	TA1535	TA1535	TA1537	TA1537	WP2uvrA	WP2uvrA	TA97	TA97	TA9B	TA98	TA100	TA100	TA102	TA102
Concentration µg/plate
0	7	8	13	7	27	28	258	265	25	33	162	132	359	398
	±4	±2	±2	±1	±8	±6	±14	±10	± 8	±10	±11	±14	± 26	± 31
8	13	7	13	8	28	29	-287	281	24	31	110	125	358	434
	±2	±1	±4	±2	±5	±3	±15	±5	± 6	± 6	±13	± 20	± 18	± 22
40	11	4	11	6	35	31	285	276	21	32	98	144	342	423
	±1	±1	±3	±2	±9	±3	±15	± 3	± 4	±9	±12	±21	± 11	± 14
200	13	8	10	8	30	32	273	295	27	35	102	107	325	343
	±3	±1	±1	±5	±4	±3	±28	±13	± 2	± 3	± 8	±8	± 20	± 17
1000	11	8	9	8	26	35	298	301	23	30	100	105	217	233
	±2	±6	±4	±3	± 6	± 3	±21	±27	± 4	± 4	± 7	± 15	± 17	± 26
5000	12	7	11	10	33	34	304	257	27	49	107	113	256	189
	±3	±3	± 4	± 5	±11	± 6	±29	±36	± 1	± 14	±10	± 10	± 38	± 19

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It can be concluded that neither AHTN itself nor any metabolite generated by the metabolic activation system is mutagenic in the Ames test under the described experimental conditions.

Executive summary:

AHTN was investigated for mutagenic potential using two versions of the Ames test: the standard plate incorporation and the preincubation methods. Seven tester strains were employed (S. typhimurium TA1535, TA1537, TA97, TA98, TA100, TA1O2 and E. coli VP2 uvrA). The cells were exposed to the test compound in absence and presence of a metabolic activation systent (S9 mix) prepared from the livers of phenobarbital/8-naphthoflavone treated rats.

Responsiveness of the strains and activity of the S9 mix were verified by including appropriate positive control compounds.

The test compound was dissolved in DMSO. Milky suspensions concentrations of 158 pg/plate and above. The dose range was chosen 5000 µg/plate. No toxicity was observed up to 5000 µg/plate.

No increase of the number of reverting colonies were observed for any of the seven tester stains.

Thus it can be concluded that neither AHTN nor any of its metabolites are mutagenic in the Ames test under the described conditions.