4-Oxazolidinone, 3-cyclohexyl-2-(cyclohexylimino)-5-[[4-(diethylamino)-2-methoxyphenyl]methylene]-

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:

2013-07-19 to 2013-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to OECD and EU guideline and in accordance with GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

FC-131

IUPAC Name:

FC-131

Constituent 2

Reference substance name:

-

EC Number:

457-890-2

EC Name:

-

Cas Number:

860399-11-7

Molecular formula:

C27H39N3O3

IUPAC Name:

(2E,5Z)-3-cyclohexyl-2-(cyclohexylimino)-5-{[4-(diethylamino)-2-methoxyphenyl]methylidene}-1,3-oxazolidin-4-one

Constituent 3

Reference substance name:

453.62 g/mol

IUPAC Name:

453.62 g/mol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report):FC-131
- Physical state: yellow crystalline powder
- Analytical purity: 99.8% (area HPLC)
- Impurities (identity and concentrations): about 0.1% 4-diethylamino-2-methoxy-benzaldehyde
- Isomers composition:No data
- Purity test date:03.07.2013
- Lot/batch No.:30610045
- Expiration date of the lot/batch:12.06.2015
- Homogeneity: yes
- Storage condition of test material: Room temperature (20+-5 °C), no humidity
- Production date: Jun. 2013

Method

Target gene:

missing Histidine synthesis

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium LT2: TA1535, TA 97a, TA98, TA 100, TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth for overnight culture (Merck 5443)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Genotype confirmation was performed once a quarter
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Additional strain / cell type characteristics:

other: all with mutation in histidine gene

Metabolic activation:

with and without

Metabolic activation system:

produced from the livers of male Sprague-Dawley rats treated with 500 mg Aroclor 1254/kg b.w. intraperitoneally

Test concentrations with justification for top dose:

nominal concentrations:
- first experiment (plate incorporation method):
5002 µg/plate , 1501 µg/plate, 502 µg/plate, 152 µg/plate and 51 µg/plate (= 50.02; 15.01 ;5.02; 15.2 and 0.51 g/L)
- second experiment (pre-incubation method):
5002 µg/plate , 2500 µg/plate, 1254 µg/plate, 624 µg/plate, 313 µg/plate, 157 µg/plate and 76 µg/plate (= 50.02; 25 ; 12.54; 6.24; 3.13; 1.57 and 0.76 g/L)

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle:
The test item was not sufficiently soluble in deionised water, dimethyl sulfoxide (DMSO) or ethanol. In DMSO, the test item showed the best homogenous suspension under permanent shaking in the highest required concentration.
Therefore, the test item was weighed directly. The weighed amounts were autoclaved and filled up with sterile DMSO.
The suspensions were shaken for 24 hours and stirred during application.
In both experiments, the two highest concentrations were tested as suspension (in the first experiment: 5002 µg/plate and 1501 µg/plate; in the second experiment: 5002 µg/plate and 2500 µg/plate).
Complete dissolution was observed at the following nominal concentrations:
Experiment I: 502 µg/plate, 152 µg/plate and 51 µg/plate.
Experiment II: 1254 µg/plate, 624 µg/plate, 313 µg/plate, 157 µg/plate and 76 µg/plate.

Controls

Untreated negative controls:

yes

Remarks:

H2O

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro- 1,2-phenylene diamine, 2-Amino-anthracene

Remarks:

NPD (TA97a, TA98, TA102; no metabolic activation); Na-azide (TA 100, TA 1535, no metabolic activation), 2AA (TA 97a, TA100, TA 102, TA 1535, +S9), BaP (TA 98, + S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
experiment 1: in agar (plate incorporation);
experiment 2: preincubation

DURATION:
- Preincubation period: 20min
- Exposure duration:48h (second experiment no data)
- Expression time (cells in growth medium): at least 1E+09 cells/ml


NUMBER OF REPLICATIONS:
4 plates for each concentration, strain, metabolic activation/no metabolic activation

NUMBER OF CELLS EVALUATED: see Table 1a (experiment 1) and Table 1b (experiment 2) below

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (The test item is considered non-toxic, if the quotient titre/tox is below 2.)

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
All calculations are performed with unrounded values.

Statistics:

calculation of mean values and standard deviations, calculation of increase factor, no documentation of significance determination

Results and discussion

Test results

Species / strain:

other: TA1535, TA97a, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test item not completely soluble in DMSO (undissolved particles have been observed) at the two highest tested concentrations (5002 and 1501 µg/plate) and were tested as suspensions in these concentrations
- Precipitation: undissolved substance particles have been observed at the highest tested concentrations


RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: only marginal differences compared to historical cotrol data

OTHER CONTROL DATA:

Genotype Confirmation: ok
Histidine Requirement: ok
UV-Sensitivity (uvrB): ok
Crystal Violet Sensitivity (deep rough/rfa): ok

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 3: Results of experiment 1 (negative controls, positive controls, values for FC-131)

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	150	125	15	15	110	123	180	184	22	22
	sd	9.9	16.4	5.9	3.6	5.1	13.6	25.2	15.2	4.4	2.2
DMSO	Mean	126	103	16	14	113	140	174	169	22	20
	sd	12.3	7.9	5.4	2.6	6.9	13.9	18.2	23.9	5.5	6.2
Positive Controls*	Mean	600	552	139	110	619	520	683	895	468	112
	sd	81	84	17	14	55	84	54	76	39	17
	f(I)	4.76	5.36	8.69	7.86	5.63	3.71	3.93	5.30	21.27	5.60
5002 µg/pl.	Mean	107	109	18	14	128	116	179	196	20	21
	sd	7	16	1	2	19	18	13	12	3	3
	f(I)	0.85	1.06	1.13	1.00	1.13	0.83	1.03	1.16	0.91	1.05
1501 µg/pl.	Mean	112	102	16	18	109	131	190	175	22	20
	sd	8	5	3	2	18	25	21	15	3	3
	f(I)	0.89	0.99	1.00	1.29	0.96	0.94	1.09	1.04	1.00	1.00
502 µg/pl.	Mean	120	121	13	13	119	136	167	178	21	23
	sd	9	20	3	2	20	20	15	19	7	4
	f(I)	0.95	1.17	0.81	0.93	1.05	0.97	0.96	1.05	0.95	1.15
152 µg/pl.	Mean	127	120	16	13	122	122	185	189	22	24
	sd	13	26	3	2	7	7	14	17	2	2
	f(I)	1.01	1.17	1.00	0.93	1.08	0.87	1.06	1.12	1.00	1.20
51 µg/pl.	Mean	112	116	13	13	116	122	193	192	24	23
	sd	21	9	3	1	19	3	19	18	3	3
	f(I)	0.89	1.13	0.81	0.93	1.03	0.87	1.11	1.14	1.09	1.15

Applicant's summary and conclusion

Conclusions:

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

Under the test conditions, the test item FC-131 is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with or without metabolic activation. Cytotoxicity of the test item at the concentrations tested was not detected.
No complete dissolution in DMSO/ Ethanol/water was possible for the two highest concentrations in the first and in the second experiment; hence undissolved particles were visible on the plates and substance was tested as suspension.

Executive summary:

The mutagenic potential of FC-131 was tested according to the OECD guideline 471 with the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 with and without metabolic activation (using S9) and applying both the plate incorporation and pre-incubation method. The tests and all included controls were valid. FC-131 was tested with a concentration range between 76 µg/plate (= 0.76 g/L) and 5002 µg/plate (= 50 g/L). Concentrations above 1501 µg/plate showed precipitation. No cytotoxicity was observed upto and including the highest concentration of 5002 µg/plate.

For all strains and all conditions tested, the test item FC-131 showed negative results and can be considered as not mutagenic under the conditions of the test.