2,2'-(vinylenedi-p-phenylene)bisbenzoxazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Ames assay was performed to investigate the potential of 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0 (NC), 0, (VC) 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0 (NC), 0 (VC), 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether (CAS no. 93-04-9) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of inhouse historical data. Whereas reference mutagens showed a distinct increase in induced revertant colonies in all the tester strains both in the presence as well as in the absence of metabolic activation without showing cytotoxicity.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Chromosome aberration:

test chemical failed to induce chromosome aberrations in the Chinese hamster ovary cells.

Hence the test chemical is not likely to classify as in-vitro gene toxicity as per the criteria mentioned in CLP regulation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-02-2018 to 30-03-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of test item 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no.1533-45-5) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0 (NC), 0 (VC), 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0, 0, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.001 – 2.5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentrations 2.5 mg/plate (T8), 0.791 mg/plate (T7) both in absence and in the presence of metabolic activation and no reduction in colony count as well as in background lawn in treated concentrations (0.250 (T6) mg/plate – 0.001 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: (0, 0, 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	108	20	109	17
	R2	110	18	113	21
	R3	114	18	117	21
VC (0.00)	R1	128	26	140	29
	R2	136	26	135	28
	R3	132	24	131	27
T1 (0.001)	R1	110	19	112	21
	R2	114	20	109	19
	R3	112	20	111	21
T2 (0.003)	R1	112	22	115	21
	R2	112	20	119	22
	R3	114	20	112	19
T3 (0.008)	R1	116	22	115	23
	R2	114	24	119	19
	R3	112	20	119	22
T4 (0.025)	R1	118	22	121	21
	R2	114	24	123	23
	R3	116	22	127	22
T5 (0.079)	R1	116	24	129	24
	R2	120	24	131	22
	R3	118	22	127	23
T6 (0.250)	R1	122	26	125	23
	R2	118	22	129	22
	R3	120	24	132	25
T7 (0.791)	R1	124	24	133	26
	R2	120	26	135	24
	R3	122	24	137	23
T8 (2.5)	R1	128	26	138	25
	R2	126	26	137	26
	R3	128	22	136	24
PC	R1	1176	1038	1352	1256
	R2	1160	1014	1376	1304
	R3	1112	992	1304	1288

NC           =     Negative control

VC           =   Vehicle Control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD
(TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	17	109	232
	R2	4	9	21	113	228
	R3	5	11	21	117	234
VC (0.00)	R1	7	14	29	140	260
	R2	7	16	28	135	248
	R3	6	12	27	131	254
T1 (0.003)	R1	5	10	21	115	230
	R2	6	12	22	119	236
	R3	5	10	19	112	238
T2 (0.008)	R1	6	12	23	115	234
	R2	4	10	19	119	240
	R3	5	12	22	119	238
T3 (0.025)	R1	6	14	21	121	236
	R2	6	10	23	123	242
	R3	5	12	22	127	240
T4 (0.079)	R1	7	14	24	129	246
	R2	6	12	22	131	238
	R3	5	12	23	127	244
T5 (0.250)	R1	7	12	23	125	252
	R2	6	14	22	129	248
	R3	6	14	25	132	242
PC	R1	188	520	1256	1352	1472
	R2	177	508	1304	1376	1560
	R3	165	492	1288	1304	1640

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	11	20	108	230
	R2	5	9	18	110	214
	R3	4	11	18	114	223
VC (0.00)	R1	7	14	26	128	248
	R2	6	12	26	136	256
	R3	8	14	24	132	252
T1 (0.003)	R1	5	10	22	112	224
	R2	5	12	20	112	224
	R3	5	12	20	114	226
T2 (0.008)	R1	5	10	22	116	228
	R2	6	14	24	114	222
	R3	6	12	20	112	226
T3 (0.025)	R1	6	13	22	118	224
	R2	5	12	24	114	228
	R3	5	10	22	116	232
T4 (0.079)	R1	6	10	24	116	226
	R2	6	12	24	120	232
	R3	6	14	22	118	236
T5 (0.250)	R1	7	12	26	122	228
	R2	6	14	22	118	234
	R3	6	12	24	120	238
PC	R1	167	1024	1038	1176	1608
	R2	179	1032	1014	1160	1656
	R3	188	1040	992	1112	1688

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                     2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                           Sodium azide [10μg/plate]: TA 1535, TA 100                                              

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	21	98	240
	R2	5	11	19	100	238
	R3	5	10	18	88	232
VC (0.00)	R1	8	15	28	117	256
	R2	7	16	26	120	248
	R3	8	17	25	123	240
T1 (0.003)	R1	5	10	19	98	236
	R2	5	11	20	100	244
	R3	6	12	20	102	240
T2 (0.008)	R1	5	10	22	106	242
	R2	6	13	21	102	238
	R3	6	12	21	100	250
T3 (0.025)	R1	6	12	23	104	248
	R2	5	12	22	106	244
	R3	7	13	20	104	246
T4 (0.079)	R1	7	13	23	105	242
	R2	6	14	24	107	248
	R3	7	13	24	104	252
T5 (0.250)	R1	7	14	26	110	248
	R2	8	15	25	106	242
	R3	7	16	24	108	250
PC	R1	178	408	1104	1408	1280
	R2	183	366	1128	1384	1352
	R3	187	378	1176	1324	1312

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	11	21	96	255
	R2	4	12	19	104	247
	R3	4	11	18	89	259
VC (0.00)	R1	7	15	27	121	272
	R2	8	16	26	117	268
	R3	8	16	22	108	260
T1 (0.003)	R1	5	11	21	100	250
	R2	4	11	19	104	254
	R3	5	12	22	106	260
T2 (0.008)	R1	5	12	21	100	260
	R2	6	11	20	104	252
	R3	5	12	22	108	258
T3 (0.025)	R1	6	10	21	106	264
	R2	6	12	22	104	254
	R3	5	14	21	104	258
T4 (0.079)	R1	6	12	23	105	260
	R2	7	14	22	104	254
	R3	6	12	23	108	266
T5 (0.250)	R1	6	14	24	106	262
	R2	8	12	22	110	268
	R3	6	14	25	112	254
PC	R1	189	1384	984	1320	1552
	R2	197	1392	888	1344	1624
	R3	192	1376	876	1240	1584

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 4 -    MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	0.58	10.00	1.00	19.67	2.31	113.00	4.00	231.33	3.06
VC (0.00)	6.67	0.58	14.00	2.00	28.00	1.00	135.33	4.51	254.00	6.00
T1 (0.003)	5.33	0.58	10.67	1.15	20.67	1.53	115.33	3.51	234.67	4.16
T2 (0.008)	5.00	1.00	11.33	1.15	21.33	2.08	117.67	2.31	237.33	3.06
T3 (0.025)	5.67	0.58	12.00	2.00	22.00	1.00	123.67	3.06	239.33	3.06
T4 (0.079)	6.00	1.00	12.67	1.15	23.00	1.00	129.00	2.00	242.67	4.16
T5 (0.250)	6.33	0.58	13.33	1.15	23.33	1.53	128.67	3.51	247.33	5.03
PC	176.67	11.50	506.67	14.05	1282.67	24.44	1344.00	36.66	1557.33	84.03

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	0.58	10.33	1.15	18.67	1.15	110.67	3.06	222.33	8.02
VC (0.00)	7.00	1.00	13.33	1.15	25.33	1.15	132.00	4.00	252.00	4.00
T1 (0.003)	5.00	0.00	11.33	1.15	20.67	1.15	112.67	1.15	224.67	1.15
T2 (0.008)	5.67	0.58	12.00	2.00	22.00	2.00	114.00	2.00	225.33	3.06
T3 (0.025)	5.33	0.58	11.67	1.53	22.67	1.15	116.00	2.00	228.00	4.00
T4 (0.079)	6.00	0.00	12.00	2.00	23.33	1.15	118.00	2.00	231.33	5.03
T5 (0.250)	6.33	0.58	12.67	1.15	24.00	2.00	120.00	2.00	233.33	5.03
PC	178.00	10.54	1032.00	8.00	1014.67	23.01	1149.33	33.31	1650.67	40.27

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

TABLE 5 -    MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD
(TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	5.00	0.00	10.33	0.58	19.33	1.53	95.33	6.43	236.67	4.16
VC (0.00)	7.67	0.58	16.00	1.00	26.33	1.53	120.00	3.00	248.00	8.00
T1 (0.003)	5.33	0.58	11.00	1.00	19.67	0.58	100.00	2.00	240.00	4.00
T2 (0.008)	5.67	0.58	11.67	1.53	21.33	0.58	102.67	3.06	243.33	6.11
T3 (0.025)	6.00	1.00	12.33	0.58	21.67	1.53	104.67	1.15	246.00	2.00
T4 (0.079)	6.67	0.58	13.33	0.58	23.67	0.58	105.33	1.53	247.33	5.03
T5 (0.250)	7.33	0.58	15.00	1.00	25.00	1.00	108.00	2.00	246.67	4.16
PC	182.67	4.51	384.00	21.63	1136.00	36.66	1372.00	43.27	1314.67	36.07

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.33	0.58	11.33	0.58	19.33	1.53	96.33	7.51	253.67	6.11
VC (0.00)	7.67	0.58	15.67	0.58	25.00	2.65	115.33	6.66	266.67	6.11
T1 (0.003)	4.67	0.58	11.33	0.58	20.67	1.53	103.33	3.06	254.67	5.03
T2 (0.008)	5.33	0.58	11.67	0.58	21.00	1.00	104.00	4.00	256.67	4.16
T3 (0.025)	5.67	0.58	12.00	2.00	21.33	0.58	104.67	1.15	258.67	5.03
T4 (0.079)	6.33	0.58	12.67	1.15	22.67	0.58	105.67	2.08	260.00	6.00
T5 (0.250)	6.67	1.15	13.33	1.15	23.67	1.53	109.33	3.06	261.33	7.02
PC	192.67	4.04	1384.00	8.00	916.00	59.19	1301.33	54.45	1586.67	36.07

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Conclusions:

Test substance did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of test substance to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0 (NC), 0, (VC) 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0 (NC), 0 (VC), 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether (CAS no. 93-04-9) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of inhouse historical data. Whereas reference mutagens showed a distinct increase in induced revertant colonies in all the tester strains both in the presence as well as in the absence of metabolic activation without showing cytotoxicity.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

WoE report is based on two in-vitro gene toxicity studies
1. In vitro mammalian chromosome aberration test was performed to determine the test chemical
2.In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-W-B1, 1

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5a medium supplemented with 10% fetal calf serum, L-glutamine,
and antibiotics.- Properly maintained: No data available- Periodically checked for Mycoplasma cont
amination: No data available- Periodically checked for karyotype stability: No data available- Peri
odically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mammalian cell line, other: Chinese hamster bone marrow cells

Remarks:

2.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consisted of 15 μl/ml liver homogenate (from male Sprague-Dawley rats, induced with Arocl or 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.

Test concentrations with justification for top dose:

1.0.04-0.8 μg/mL
2.500, 1000 or 2000 mg/Kg

Vehicle / solvent:

1.- Vehicle(s)/solvent(s) used: Ethanol - Justification for choice of solvent/vehicle: The chemical was soluble in ethanol.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Triethylenemelamine

Remarks:

1.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

2.

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: In mediumDURATION- Preincubation period: No data available- Exposure duration: Cells were exposed to the test chemical for 2 hr in the presence of S9 or for 20 hrs without S9.-
Expression time (cells in growth medium): 28.5 – 37.3 hrs during the delayed harvest time- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): The cell harvest time for the aberration test was 8-12 hr after the beginning of treatme nt. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 28.5 – 37.3 hr, were used to allow delayed cells to reach mitosis.SELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: No data availableNUMBER OF CELLS
EVALUATED: 100 cells were scored from each of the three highest dose groups having sufficient meta phases for analysisDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No dataOTHER: No data available.

2. METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: No data
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other:
OTHER: No data

Evaluation criteria:

1. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploidy cells but used metaphases with 19-23 chromosomes (the modal number being 21).

2. The cell line was observed for chromatid breaks, chromatid exchanges, acentric fragments, pulverisations, etc. and anomolous interphase cells

Statistics:

1. Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-W-B1, 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: Chinese hamster bone marrow cells

Remarks:

2.

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available
- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No available data
RANGE-FINDING/SCREENING
STUDIES: Dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: Non mutagenic

Conclusions:

2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5)does not exhibit gene toxicity toxicity.

Executive summary:

Chromosome aberration:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5). When administered to Chinese Hamster Ovary (CHO) cells. 

Study 1:

An in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical using Chinese hamster ovary cells (CHO-W-B1) at dose levels of 0.04- 0.8 µg/mL. Cells were exposed to the test chemical for 2 hr in the presence of S9 or for 20hrs without S9 and were incubated for 28.5 – 37.3 hrs during the delayed harvest time. One hundred cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. Based on the results noted, test chemical failed to induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system, and hence is not classified as a gene mutant in vitro.

Study 2:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster bone marrow cells. The test chemical was tested at dose levels of 500, 1000 or 2000 mg/Kg and cyclophosphamide at 128 mg/kg was used as the positive control chemical. The treated cell line wad scored for aberrant metaphases (chromatid break, chromatid exchanges, acentric fragments, pulverisations, etc.) and anomolous interphase cells. The test chemical did not cause a significant enhancement of the aberrant metaphases or of anomalous interphase cells in the Chinese hamster bone marrow cells and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5)does not exhibit gene toxicity toxicity. Hence the test chemical is not likely to classify as in-vitro gene toxicity as per the criteria mentioned in CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data availabe for the target chemical and from the two read across have been reviewed to determine the mutagenic nature of 2,2'-(Vinylenedi-4-phenylene)bis(benzoxazole) (CAS no 1533 -45 -5; IUPAC name: 2,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole)). The studies are as mentioned below:

Ames assay was performed (Sustainability Support Services (Europe) AB, 2018) to investigate the potential of 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0 (NC), 0, (VC) 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0 (NC), 0 (VC), 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl-2-napthyl ether (CAS no. 93-04-9) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of inhouse historical data. Whereas reference mutagens showed a distinct increase in induced revertant colonies in all the tester strains both in the presence as well as in the absence of metabolic activation without showing cytotoxicity. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Based on the data available for the target chemical, 2,2'-(Vinylenedi-4-phenylene)bis(benzoxazole) (CAS no 1533 -45 -5) is not likely to exhibit genetic toxicity in vitro. Thus, the chemical is not classified as a genetic toxicant as per as per the criteria mentioned in CLP regulation.

Chromosome aberration:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5). When administered to Chinese Hamster Ovary (CHO) cells. 

Study 1:

An in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical using Chinese hamster ovary cells (CHO-W-B1) at dose levels of 0.04- 0.8 µg/mL. Cells were exposed to the test chemical for 2 hr in the presence of S9 or for 20hrs without S9 and were incubated for 28.5 – 37.3 hrs during the delayed harvest time. One hundred cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. Based on the results noted, test chemical failed to induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system, and hence is not classified as a gene mutant in vitro.

Study 2:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster bone marrow cells. The test chemical was tested at dose levels of 500, 1000 or 2000 mg/Kg and cyclophosphamide at 128 mg/kg was used as the positive control chemical. The treated cell line wad scored for aberrant metaphases (chromatid break, chromatid exchanges, acentric fragments, pulverisations, etc.) and anomolous interphase cells. The test chemical did not cause a significant enhancement of the aberrant metaphases or of anomalous interphase cells in the Chinese hamster bone marrow cells and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5)does not exhibit gene toxicity toxicity. Hence the test chemical is not likely to classify as in-vitro gene toxicity as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical, 2,2'-(Vinylenedi-4-phenylene)bis(benzoxazole) (CAS no 1533 -45 -5) is not likely to exhibit genetic toxicity in vitro. Thus, the chemical is not classified as a genetic toxicant as per as per the criteria mentioned in CLP regulation.