2,2,5-trimethyl-5-pentylcyclopentan-1-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From June 10 to August 26, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted according to OECD test Guideline No. 476 without any deviation. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on March 4 to 8, 2013/ signed on May 6, 2013

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-pentylcyclopentan-1-one

EC Number:

225-392-2

EC Name:

2-pentylcyclopentan-1-one

Cas Number:

4819-67-4

IUPAC Name:

2-pentylcyclopentanone

Test material form:

other: liquid

Details on test material:

- Physical state: Clear colourless liquid
- Storage condition of test material: At room temperature in the dark in aluminium bottle

Method

Target gene:

TK+/- gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically "cleansed" against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (4 % v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats orally induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Dose range finding test: 17, 52, 164, 512 and 1545 μg/mL, in the absence of S9-mix with a 3 and 24 h treatment period and in the presence of S9-mix with a 3 h treatment period
First mutagenicity test:
- Without S9-mix (3 h exposure): 0.17, 0.54, 1.7, 5.4, 17, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium
- With S9-mix (3 h exposure): 17, 52, 70, 90, 110, 130, 150, 170, 190 and 200 μg/mL exposure medium
Second mutagenicity test:
- Without S9-mix (24 h exposure): 0.54, 1.7, 5.4, 17, 30, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test substance preparation: No correction was made for the purity/composition of the test substance. The test item was immiscible in RPMI 1640 medium at a concentration of 155 mg/mL, but was fully miscible in DMSO at the required concentration, as determined in solubility checks. The test substance was therefore dissolved in DMSO (SeccoSolv, Merck Darmstadt, Germany). Test item concentrations were used within 1.5 h after preparation. The final concentration of the solvent in the exposure medium was 1.0% (v/v).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix: 15 and 5 μg/mL for a 3 and 24 h treatment period, respectively

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix: 7.5 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
Exposure medium: For 3 h exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 h exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

DURATION
- Exposure duration: Dose range finding test: 3 h (±-S9-mix); 24 h (- S9-mix); First mutagenicity test: 3 h (±-S9-mix); Second mutagenicity test: 24 h (- S9-mix)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 or 12 days
- All incubations were carried out in a humid atmosphere (80 - 100%, actual range 50 – 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.6–37.7 °C).

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Dose range finding test: single cultures/dose for test item and vehicle control; mutagenicity test: single cultures/dose for test item and positive control; duplicate cultures for vehicle control

NUMBER OF CELLS EVALUATED:
- For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium. For determination of the MF a total number of 9.6 x 10^5 cells/concentration were plated in five
96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). In the treatment group of 0.17 μg/mL in the first experiment (absence of S9-mix) a total number of 464 wells was used for determination of the mutation frequency.
- Stain: The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 h, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency (CE), Relative survival (RS), Relative Total Growth (RTG), Suspension Growth (SG), Relative Suspension Growth (RSG)

Evaluation criteria:

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF (mean solvent control) + 126.
In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Statistics:

No data

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Since the test substance precipitated in the exposure medium at concentrations of 512 μg/mL and above, the pH and osmolality were not determined.
- Precipitation: Although test item precipitated in the exposure medium at concentrations of 512 μg/mL and above, the test item could be tested up to and including the concentration of 1545 μg/mL (= 0.01 M, recommended maximum dose level in the guidelines).

DOSE RANGE FINDING TEST:
- In the absence of S9-mix (3 h treatment), no toxicity in the relative suspension growth was observed up to test substance concentrations of 52 μg/mL compared to the solvent control. Hardly any cell survival was observed at test substance concentrations of 164 μg/mL and above.
- In the presence of S9-mix (3 h treatment), the relative suspension growth was 25% at the test substance concentration of 164 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 512 μg/mL and above.
- In the absence of S9-mix (24 h treatment), the relative suspension growth was 67% at the test substance concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 164 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The spontaneous mutation frequencies in the solvent-treated control cultures were within the minimum and maximum value of the historical control data range. Historical control data generated from experiments performed between May 2011 to May 2014.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test:
- In the absence of S9-mix, the dose levels of 52 to 90 μg/mL showed similar cytotoxicity. Therefore, the dose level of 90 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 130 and 150 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 0.17, 0.54, 1.7, 5.4, 17, 52, 70 and 110 μg/mL exposure medium.
- In the presence of S9-mix, too many dose levels showed severe cytotoxicity, this part of the experiment was repeated (experiment 1A): the following dose range was selected: 5.4, 17, 52, 70, 100, 110, 120, 130, 140 and 150 μg/mL. The dose levels of 140 and 150 μg/mL were too toxic for further testing. Therefore, the dose level of 140 and 150 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels selected to measure mutation frequencies at the TK-locus were: 5.4, 17, 52, 70, 100, 110, 120 and 130 μg/mL exposure medium.
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 83% compared to the total growth of the solvent controls.

Second mutagenicity test:
- The dose levels of 0.54 to 17 μg/mL showed similar cytotoxicity. Therefore, the dose level of 0.54 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 130 and 150 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 1.7, 5.4, 17, 30, 52, 70, 90 and 110 μg/mL exposure medium.
- In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 84% compared to the total growth of the solvent controls.

Remarks on result:

other: strain/cell type: L5178Y/TK+/--3.7.2C mouse lymphoma cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Evaluation of the mutagenicity:

First mutagenicity test: No significant increase in the mutation frequency (MF < MF_{mean solvent control}+ GEF) at the TK locus was observed after treatment with test item either in the absence (MF < 236 x 10^-6) or in the presence of S9-mix (MF < 205 x 10^-6). The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test: No significant increase in the mutation frequency at the TK locus was observed after treatment with test item (MF < 239 x 10^-6). The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Table 7.6.1/1: Cytotoxic and mutagenic response of test item in the mouse lymphoma L5178Y test system

 

Dose (μg/mL)	RSG (%)	CEday2 (%)	RSday2	RTG (%)	mutation frequency per 10^6survivors
					Total	small	large
Experiment 1: Without metabolic activation - 3 h treatment
DMSO	100	80	100	100	104	37	63
DMSO		94			115 (110^1)	40	68
0.17	88	95	109	97	101	39	57
0.54	64	77	88	57	99	46	49
1.7	108	76	87	94	104	54	46
5.4	182	66	76	138	92	40	49
17	126	76	87	110	90	53	34
52	50	81	93	47	92	42	46
70	37	88	100	37	103	45	54
110	13	76	87	11	83	35	46
MMS	53	47	54	29	1050	586	343
Experiment 1: With metabolic activation - 3 h treatment
DMSO	100	88	100	100	79	24	52
DMSO		93			79 (79^1)	29	47
5.4	103	98	109	112	66	17	47
17	91	99	110	101	80	26	52
52	86	99	110	95	70	32	35
70	73	91	101	74	90	32	55
100	74	111	124	92	64	24	38
110	54	101	112	61	55	25	28
120	46	108	120	55	88	40	43
130	17	90	100	17	92	25	64
CP	73	71	79	58	633	291	271
Experiment 2: Without metabolic activation - 24 h treatment
DMSO	100	81	100	100	112	76	31
DMSO		86			114 (113^1)	82	28
1.7	91	81	97	89	121	79	37
5.4	91	77	92	84	149	98	43
17	81	80	96	77	137	92	39
30	64	83	99	63	124	84	35
52	56	95	114	63	108	83	21
70	34	80	96	33	100	74	22
90	23	75	89	20	94	61	30
110	15	86	103	16	104	66	34
MMS	96	64	77	74	806	560	168

 

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth;

MMS = Methylmethanesulfonate; CP = Cyclophosphamide

1) Mean no of mutants in the solvent control groups {SC1 + SC2}/2

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/-(3.7.2C) mouse lymphoma cells were exposed to test item at the following concentrations:

Dose range finding test: 17, 52, 164, 512 and 1545 μg/mL, in the absence of S9-mix with a 3 and 24 h treatment period and in the presence of S9-mix with a 3 h treatment period

First mutagenicity test:

- Without S9-mix (3 h exposure): 0.17, 0.54, 1.7, 5.4, 17, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium

- With S9-mix (3 h exposure): 17, 52, 70, 90, 110, 130, 150, 170, 190 and 200 μg/mL exposure medium

Second mutagenicity test:

- Without S9-mix (24 h exposure): 0.54, 1.7, 5.4, 17, 30, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium 

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 4 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone.

 

In dose range finding test, hardly any cell survival was observed at test substance concentrations of 164 μg/mL and above in the absence of S9-mix and at test substance concentrations of 512 μg/mL and above in the presence of S9-mix. In the first experiment, test item was tested up to concentrations of 110 and 130 μg/mL in the absence and presence of S9-mix, respectively. Relative total growth (RTG) was reduced to 11 and 17% in the absence and presence of S9-mix, respectively. In the second experiment, test item was tested up to concentrations of 110 μg/mL in the absence S9-mix. The RTG was reduced to 16%.

 

In the absence of S9-mix, test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, test item did not induce a significant increase in the mutation frequency in the first experiment. The mutation frequencies in the vehicle and positive control cultures were within the acceptable range indicating the validity of the study.

 

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).