2-(3,7-dimethylocta-2,6-dienyl)cyclopentan-1-one

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:

December 13, 2018 - January 18, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(3,7-dimethylocta-2,6-dienyl)cyclopentan-1-one

EC Number:

268-706-3

EC Name:

2-(3,7-dimethylocta-2,6-dienyl)cyclopentan-1-one

Cas Number:

68133-79-9

Molecular formula:

C15H24O

IUPAC Name:

2-(3,7-dimethylocta-2,6-dienyl)cyclopentan-1-one

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, R factor

Metabolic activation:

with

Metabolic activation system:

S9 derived from male Sprague-Dawley rats (phenobarbital and benzoflavone induced rat liver).

Test concentrations with justification for top dose:

0.8, 1.58, 5.0, 8, 15.8, 50, 80, 158, 500, 1580, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: ICR 191 Acridine

Controls 2

Positive controls:

yes

Positive control substance:

other: Daunomycin

Controls 3

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

Plate incorporation mutation assay:
The initial test followed the plate incorporation method, in which the following materials were mixed and poured over the surface of a minimal agar plate:
• 100 µL of the prepared test substance solutions, negative (vehicle) control, or prepared positive control substance
• 500 µL S9 mix or substitution buffer
• 100 µL bacteria suspension (ST or EC)
• 2000 µL overlay agar maintained at approximately 45°C
Plates were prepared in triplicate at each experimental point and uniquely identified. After pouring, plates were placed on a level surface until the agar gelled then inverted and incubated at approximately 37 degrees C until growth was adequate for enumeration (approximately 65 hours). Appropriate sterility control check plates (treated with critical components in the absence of bacteria) were included as a standard procedural check.

Pre-incubation assay:
The confirmatory test employed the pre-incubation modification of the plate incorporation test. The test or control substances, bacteria suspension, and S9/substitution buffer were incubated under agitation for approximately 30 minutes at approximately 37°C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The study design for the confirmatory test, including strains, dose levels etc. was as described above for the initial (main) test.

A supplemental test was performed to clarify results obtained with TA98 and TA1537 in the initial phase of testing. The test utilized the same procedures as the main test with the additional dose levels listed below. Appropriate vehicle and positive controls were included.

Evaluation criteria:

For each experimental point, the Mutation Factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:
The results were considered positive (i.e., indicative of mutagenic potential) if:
• The results for the test item showed a substantial increase in revertant colony counts, i.e., response MF ≥ 2 for strains TA98, TA100, and WP2 uvrA or MF ≥ 3 for strains TA1535 and TA1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.
• The above increase must be dose related and/or reproducible, i.e., increases must be obtained at more than one experimental point (at least one strain, more than one dose level, more than one occasion or with different methodologies).
If the second criterion is not met, the results may be classified as equivocal, and further testing may be appropriate.
A test substance that produces neither a concentration related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies is considered to be non-mutagenic in this test system.

Statistics:

Product Safety Labs calculated means and standard deviations for all quantitative data collected.

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:

cytotoxicity

Remarks:

At ≥ 500 µg/plate with S9 and ≥ 1580 µg/plate without S9 in the pre-incubation method.

Vehicle controls validity:

valid

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:

cytotoxicity

Remarks:

At ≥ 500 µg/plate with and/or without S9 in the plate incorporation method and ≥ 80 µg/plate with and/or without S9 in the pre-incubation method.

Vehicle controls validity:

valid

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:

cytotoxicity

Remarks:

At 5000 µg/plate with S9 in the plate incorporation method and at ≥ 500 µg/plate with S9 in the pre-incubation method.

Vehicle controls validity:

valid

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:

cytotoxicity

Remarks:

At ≥ 1580 µg/plate with S9 in the plate incorporation method and ≥ 80 µg/plate with and/or without S9 in the pre-incubation method.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No
- Other confounding effects: Individual plate contamination which obscured the count was noted at 500 µg/plate for strain TA 98 in the pre-incubation method without S9.

Any other information on results incl. tables

Mutation Factors

TA 98	Main Test	Main Test	Confirmatory Test	Confirmatory Test
	-S9	+S9	-S9	+S9
DMSO	1.00	1.00	1.00	1.00
1.58 ug/plate	0.85	1.04	1.00	1.20
5 ug/plate	0.89	1.07	1.04	1.04
15.8 ug/plate	0.81	1.11	1.00	0.96
50 ug/plate	1.11	1.04	1.04	1.04
158 ug/plate	0.85	0.86	0.92	0.74
500 ug/plate	0.89	0.96	1.00	1.08
1580 ug/plate	0.96	0.89	0.92	0.96
5000 ug/plate	0.81	0.93	0.75	0.88
Daunomycin 6 ug/plate	52.15	--	20.63	--
2-AA 10 ug/plate	--	155.82	--	124.48
TA100
DMSO	1.00	1.00	1.00	1.00
1.58 ug/plate	0.96	1.01	0.96	0.96
5 ug/plate	0.95	0.97	0.98	0.93
15.8 ug/plate	0.96	1.01	0.96	0.96
50 ug/plate	0.96	0.98	0.89	0.96
158 ug/plate	0.90	0.90	0.87	0.91
500 ug/plate	0.88	0.90	0.89	0.88
1580 ug/plate	0.88	0.90	0.88	0.89
5000 ug/plate	0.86	0.86	0.85	0.86
Sodium Azide 1.5 ug/plate	8.14	--	8.44	--
2-AA 10 ug/plate	--	36.17	--	38.73
TA1535
DMSO	1.00	1.00	1.00	1.00
1.58 ug/plate	0.86	0.85	0.86	0.85
5 ug/plate	0.79	0.92	1.00	1.00
15.8 ug/plate	0.93	1.00	0.86	0.77
50 ug/plate	0.93	1.00	0.79	0.85
158 ug/plate	0.79	0.62	0.71	0.77
500 ug/plate	0.79	0.85	0.79	0.69
1580 ug/plate	0.79	0.69	0.71	0.69
5000 ug/plate	0.64	0.69	0.71	0.69
Sodium Azide 1.5 ug/plate	57.79	--	58.00	--
2-AA 10 ug/plate	--	29.08	--	25.08
TA1537
DMSO	1.00	1.00	1.00	1.00
1.58 ug/plate	0.92	0.82	0.82	1.00
5 ug/plate	0.83	1.00	0.82	1.00
15.8 ug/plate	0.75	0.91	0.91	0.90
50 ug/plate	0.75	1.00	0.73	0.90
158 ug/plate	0.83	0.73	0.82	0.80
500 ug/plate	0.83	0.82	0.73	0.90
1580 ug/plate	0.75	0.73	0.73	0.90
5000 ug/plate	0.75	0.73	0.82	0.90
ICR 191 Acridine 1 ug/plate	336.08	--	702.27	--
2-AA 10 ug/plate	--	42.09	--	36.70
E. coli WP2 uvrA
DMSO	1.00	1.00	1.00	1.00
1.58 ug/plate	0.93	0.91	1.00	1.02
5 ug/plate	0.88	1.23	1.07	0.88
15.8 ug/plate	0.84	0.86	1.09	0.75
50 ug/plate	0.98	0.98	0.91	0.79
158 ug/plate	0.81	0.89	0.87	0.90
500 ug/plate	0.81	1.14	0.96	0.88
1580 ug/plate	0.98	1.00	0.69	0.75
5000 ug/plate	1.02	1.07	0.76	0.69
MMS 2.5 ug/plate	15.33	--	9.67	--
2-AA 10 ug/plate	--	2.43	--	2.00

Applicant's summary and conclusion

Conclusions:

Apritone is not mutagenic.

Executive summary:

In a reverse gene mutation test in bacteria, strains TA98, TA100, TA1535, and TA1537 of Salmonella. Typhimurium and Escherichia coli WP2 uvrA (E. coli, EC) were exposed to 2-(3,7-dimethylocta-2,6-dienyl)cyclopentan-1-one; APRITONE (98.8% purity), using dimethylsulfoxide (DMSO) solvent at concentrations of 0.8, 1.58, 5.0, 8, 15.8, 50, 80, 158, 500, 1580, and 5000mg/plate in the presence and absence of S9 activation. Three plates were used, per dose, per condition.

There was no evidence of precipitation present for any of the strains at the tested concentrations. Individual plate contamination which obscured the count was noted at 500 µg/plate for strain TA 98 in the
pre-incubation method without S9. Toxicity with presence of incomplete lawn was seen for strains TA1535, TA1537, TA98 and TA100 at ≥ 80 µg/plate with and/or without S9 in the plate incorporation and/or pre-incubation method.

For all strains, at least five non-toxic dose levels were evaluated; therefore bacterial mutagenicity was adequately assessed.

In conclusion, based on these findings and on the evaluation system used,2-(3,7-dimethylocta-2,6-dienyl)cyclopentan-1-one; APRITONEdid not elicit evidence ofbacterial mutagenicity in the Ames assay.