4-benzyloxy-2-nitro-beta-pyrrolidinostyrene

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:

From 25 April 2017 to 5 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Cas Number:

99474-22-3

Molecular formula:

C19H20N2O3

IUPAC Name:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by CHIMEX SAS batch No. 0159115
- Expiration date of the lot/batch: 21 February 2018
- Purity test date: 21 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (18.0 – 22.5°C) protected from light.
- Stability under test conditions: H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
- Solubility and stability of the test substance in the solvent/vehicle: H2O: unknown; EtOH: unknown; acetone: >1 g/L; CH3CN: unknown; DMSO: unknown
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
A stock solution containing 50 g/L of the test item in DMSO was freshly prepared on the day of the first and the second experiment. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: HisG46, uvrB, rfa

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: hisG428, pKM101, rfa, pAQ1

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa, pKM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: hisD3052, uvrB, rfa, pKM101

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA 97a

Additional strain / cell type characteristics:

other: hisD6610, uvrB, rfa, pKM101

Metabolic activation:

with and without

Metabolic activation system:

S9 obtained from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg bw intraperitoneally

Test concentrations with justification for top dose:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, ethanol and dimethyl sulfoxide (DMSO).
DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
A stock solution containing 50 g/L of the test item in DMSO was freshly prepared on the day of the first and the second experiment. The test item solution was not sterile filtrated before use.
The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.

The following nominal concentrations were prepared for the second experiment:
5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate, 156 µg/plate and 78 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Remarks:

Demineralized water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

benzo(a)pyrene

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) in experiment 1; preincubation in experiment 2
- Cell density at seeding (if applicable):10E9 bacteria/mL

DURATION
- Preincubation period: 20 minutes (in experiment 2)
- Exposure duration:48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): revertant bacterias were counted after the 48 hours exposure period

NUMBER OF REPLICATIONS: triplicates per bacteria strain, per concentration, without and with S9

DETERMINATION OF CYTOTOXICITY
- Method: bacteria background lawn
- Any supplementary information relevant to cytotoxicity: The test item is considered non-toxic if the quotient titre/toxicity is below 2.

Evaluation criteria:

The mean values and standard deviations of each threefold determination are calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 or 3 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity for a global interpretation of results.
The study is considered valid if at least five analyzable concentrations are presented in all strains of the main tests.

Statistics:

No statistical analysis of results will be done and historical data are considered to improve the interpretation of results.

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:

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 97

Remarks:

TA 97a

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

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

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:

Experiment 1 :
In the first experiment, the test item showed no precipitate on the plates in any tested concentration. No sign of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Experiment 2 :
In the second experiment, the test item showed no precipitate on the plates in all tested concentrations. No sign of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Remarks on result:

other: For the two experiments

Any other information on results incl. tables

Table2 :Summary of the results of the experiment 1

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	102	78	11	20	120	121	324	291	24	24
	sd	12.8	6.4	0.6	2.5	4.0	8.1	28.8	48.7	3.1	4.6
DMSO	Mean	88	86	13	15	115	99	335	311	31	18
	sd	15.6	24.5	2.3	2.5	18.8	9.5	77.2	87.3	2.1	5.2
Positive Controls*	Mean	580	379	407	68	441	1176	1371	1416	345	155
	sd	53.8	191.8	54.3	7.9	151.4	374.2	204.6	140.6	33.5	22.0
	f(I)	6.59	4.41	31.31	4.53	3.68	11.88	4.09	4.55	14.38	8.61
5000 µg/plate	Mean	98	91	11	10	100	105	259	331	17	11
	sd	13.9	24.3	0.0	0.6	12.5	16.5	71.8	25.7	1.5	3.2
	f(I)	1.11	1.06	0.85	0.67	0.87	1.06	0.77	1.06	0.55	0.61
1500 µg/plate	Mean	85	82	14	11	96	110	324	337	18	24
	sd	5.3	13.9	2.3	2.9	11.0	13.3	64.4	102.9	1.7	2.5
	f(I)	0.97	0.95	1.08	0.73	0.83	1.11	0.97	1.08	0.58	1.33
500 µg/plate	Mean	86	81	13	16	101	96	311	263	22	16
	sd	5.6	10.5	2.1	2.6	6.0	20.4	40.5	16.2	5.0	3.6
	f(I)	0.98	0.94	1.00	1.07	0.88	0.97	0.93	0.85	0.71	0.89
150 µg/plate	Mean	88	70	15	15	108	81	277	301	24	24
	sd	6.4	17.1	1.0	5.5	21.1	4.4	36.9	63.6	1.2	5.5
	f(I)	1.00	0.81	1.15	1.00	0.94	0.82	0.83	0.97	0.77	1.33
50 µg/plate	Mean	74	75	16	14	89	79	344	360	28	24
	sd	6.8	13.3	3.6	3.6	1.0	4.0	59.2	38.2	1.5	6.8
	f(I)	0.84	0.87	1.23	0.93	0.77	0.80	1.03	1.16	0.90	1.33

 

Table3 :Summary of the results of the experiment 2

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	84	78	18	13	95	102	313	281	15	13
	sd	11.8	4.7	2.6	1.5	16.6	12.1	95.9	58.0	2.5	3.5
DMSO	Mean	85	95	17	33	81	86	320	315	15	10
	sd	12.5	12.9	2.5	34.1	10.2	9.0	52.3	70.5	2.5	1.5
Positive Controls*	Mean	389	364	407	237	628	835	1504	1267	111	81
	sd	77.2	94.6	72.6	18.5	101.0	264.9	92.3	234.1	21.6	7.6
	f(I)	4.58	3.83	23.94	7.18	6.61	9.71	4.70	4.02	7.40	8.10
5000 µg/plate	Mean	73	70	12	14	79	97	293	237	12	8
	sd	7.6	10.6	1.5	1.0	6.2	12.2	41.6	62.1	2.0	1.2
	f(I)	0.86	0.74	0.71	0.42	0.98	1.13	0.92	0.75	0.80	0.80
2500 µg/plate	Mean	79	81	14	13	77	105	227	241	13	8
	sd	17.6	4.9	2.5	1.0	9.6	18.6	23.1	28.9	3.6	1.2
	f(I)	0.93	0.85	0.82	0.39	0.95	1.22	0.71	0.77	0.87	0.80
1250 µg/plate	Mean	73	86	15	16	94	107	288	208	10	9
	sd	11.8	3.5	6.4	7.8	7.8	21.2	24.3	94.1	0.6	0.6
	f(I)	0.86	0.91	0.88	0.48	1.16	1.24	0.90	0.66	0.67	0.90
625 µg/plate	Mean	74	75	12	14	100	119	259	271	13	15
	sd	13.3	4.5	1.7	1.2	22.6	5.0	12.2	42.0	3.1	3.2
	f(I)	0.87	0.79	0.71	0.42	1.23	1.38	0.81	0.86	0.87	1.50
312 µg/plate	Mean	64	96	16	15	95	102	290	325	11	18
	sd	2.5	23.8	1.2	0.6	15.0	15.9	45.3	84.3	1.5	5.3
	f(I)	0.75	1.01	0.94	0.45	1.17	1.19	0.91	1.03	0.73	1.80
156 µg/plate	Mean	68	73	14	17	83	105	320	241	12	17
	sd	10.6	8.2	4.0	3.1	7.5	15.0	52.0	76.9	2.5	4.9
	f(I)	0.80	0.77	0.82	0.52	1.02	1.22	1.00	0.77	0.80	1.70
78 µg/plate	Mean	90	89	15	26	89	112	261	313	13	13
	sd	17.2	14.7	1.5	2.9	12.7	29.1	55.0	75.6	2.9	3.1
	f(I)	1.06	0.94	0.88	0.79	1.10	1.30	0.82	0.99	0.87	1.30

 

Table4 :Historical data of spontaneousrevertants

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Demin. H2O	Mean	94	98	14	17	94	97	276	296	17	16
	Min	60	63	6	8	62	66	85	67	6	7
	Max	144	138	35	41	141	141	425	511	30	30
	SD	20	16	5	5	15	15	68	80	6	6
	Exp1	102	78	11	20	120	121	324	291	24	24
	Exp2	84	78	18	13	95	102	313	281	15	13
DMSO	Mean	92	102	13	15	91	94	276	288	16	16
	Min	58	70	7	8	60	63	79	80	8	6
	Max	135	144	46	36	136	199	393	459	33	29
	SD	19	17	5	5	16	20	63	69	6	6
	Exp1	88	86	13	15	115	99	335	311	31	18
	Exp2	85	95	17	33	81	86	320	315	15	10
Positive Controls*	Mean	561	507	407	73	514	727	1133	1242	251	117
	Min	264	241	112	39	223	273	491	408	55	45
	Max	1152	1181	793	217	984	1912	2331	6083	484	712
	SD	174	149	141	30	154	296	435	708	90	84
	Exp1	580	379	407	68	441	1176	1371	1416	345	155
	Exp2	389	364	407	237	628	835	1504	1267	111	81

 

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the study, the registered item Imexine OBA/50 SEC revealed no cytotoxic effrect nor genotoxicity in the bacteria strains used. Hence, it was considered as non-mutagenic for bacteria.

Executive summary:

This GLP-compliant study was performed in order to evaluate the mutagenic potential of IMEXINE OBA/50 SEC in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium.

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation. In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Based on the first experiment, the test item was tested up to concentrations of 5000  µg/plate in the absence and presence of S9-mix in all bacteria strain using the pre-incubation method. The test item showed no precipitate on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in any bacteria strain. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in any bacteria strain compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in any strain, in the presence and absence of metabolic activation.

Under the experimental conditions of the study, the registered item Imexine OBA/50 SEC revealed no cytotoxic effrect nor genotoxicity in the bacteria strains used. Hence, it was considered as non-mutagenic for bacteria.