Methyl 5-nitrohydrogen.isophthalate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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 the test chemical 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:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 5-nitrohydrogen.isophthalate

EC Number:

217-793-6

EC Name:

Methyl 5-nitrohydrogen.isophthalate

Cas Number:

1955-46-0

Molecular formula:

C9H7NO6

IUPAC Name:

3-(methoxycarbonyl)-5-nitrobenzoic acid

Method

Target gene:

Histidine

Species / strain

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, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate

Vehicle / solvent:

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

Controls

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:

Mean was observed.

Results and discussion

Test results

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 (0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
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) 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: (0.050, 0.158, 0.501, 1.582 and 5 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

Any other information on results incl. tables

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	4	10	21	110	236
	R2	5	11	20	114	242
	R3	4	10	22	118	260
VC (0.00)	R1	7	14	26	126	282
	R2	6	16	25	124	272
	R3	7	13	26	128	280
T1 (0.005)	R1	5	10	23	114	246
	R2	4	12	22	116	262
	R3	5	11	20	118	254
T2 (0.016)	R1	5	12	23	114	266
	R2	6	10	22	116	260
	R3	6	14	21	120	252
T3 (0.050)	R1	5	12	23	118	274
	R2	6	10	22	112	260
	R3	5	12	23	116	268
T4 (0.158)	R1	7	14	21	121	280
	R2	6	14	24	116	266
	R3	5	12	25	118	288
T5 (0.501)	R1	6	14	22	122	278
	R2	6	15	24	120	266
	R3	7	10	20	114	272
PC	R1	188	504	1380	1704	1408
	R2	168	488	1348	1620	1512
	R3	152	516	1312	1664	1448

 

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	4	10	20	102	210
	R2	4	11	18	106	218
	R3	4	11	21	104	212
VC (0.00)	R1	6	16	24	120	286
	R2	7	12	21	114	282
	R3	7	14	22	118	280
T1 (0.005)	R1	4	12	19	104	238
	R2	5	12	22	102	256
	R3	5	10	21	106	240
T2 (0.016)	R1	5	12	20	116	252
	R2	5	11	22	114	248
	R3	5	10	18	113	230
T3 (0.050)	R1	5	12	19	111	256
	R2	6	14	22	109	260
	R3	6	12	23	112	274
T4 (0.158)	R1	7	12	21	117	252
	R2	6	13	18	111	266
	R3	6	12	22	99	264
T5 (0.501)	R1	5	12	20	106	280
	R2	6	14	19	120	272
	R3	5	14	23	115	280
PC	R1	156	1088	880	1592	1520
	R2	172	1160	900	1448	1472
	R3	140	1120	960	1520	1536

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	4	13	20	114	230
	R2	5	10	20	112	252
	R3	5	12	18	108	242
VC (0.00)	R1	7	16	25	124	268
	R2	6	14	24	132	272
	R3	8	15	24	128	268
T1 (0.005)	R1	6	14	19	112	250
	R2	4	12	22	120	240
	R3	5	11	21	116	238
T2 (0.016)	R1	5	12	18	114	244
	R2	4	12	22	124	240
	R3	4	15	20	118	242
T3 (0.050)	R1	6	15	23	122	250
	R2	6	13	20	126	256
	R3	7	14	22	118	254
T4 (0.158)	R1	7	14	23	116	248
	R2	5	13	20	120	240
	R3	6	14	18	114	244
T5 (0.501)	R1	7	15	24	126	268
	R2	7	13	22	122	252
	R3	6	14	25	124	266
PC	R1	186	432	1296	1472	1512
	R2	164	388	1216	1520	1472
	R3	190	380	1192	1552	1552

 

 

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	4	13	20	112	234
	R2	4	12	21	110	246
	R3	5	10	19	114	240
VC (0.00)	R1	7	15	24	126	270
	R2	6	15	26	128	264
	R3	7	14	22	122	268
T1 (0.005)	R1	5	13	21	114	238
	R2	5	11	21	120	242
	R3	4	12	20	118	244
T2 (0.016)	R1	7	12	23	112	236
	R2	5	12	20	114	236
	R3	6	13	23	112	254
T3 (0.050)	R1	6	13	20	118	256
	R2	7	12	21	120	244
	R3	6	13	23	122	258
T4 (0.158)	R1	7	14	24	120	244
	R2	6	11	20	121	248
	R3	5	12	19	118	242
T5 (0.501)	R1	7	15	24	124	250
	R2	6	12	23	118	268
	R3	7	14	23	120	254
PC	R1	192	1240	772	1144	1616
	R2	188	1168	804	1216	1552
	R3	202	1296	928	1248	1624

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)	4.33	0.58	10.33	0.58	21.00	1.00	114.00	4.00	246.00	12.49
VC (0.00)	6.67	0.58	14.33	1.53	25.67	0.58	126.00	2.00	278.00	5.29
T1 (0.005)	4.67	0.58	11.00	1.00	21.67	1.53	116.00	2.00	254.00	8.00
T2 (0.0.016)	5.67	0.58	12.00	2.00	22.00	1.00	116.67	3.06	259.33	7.02
T3 (0.050)	5.33	0.58	11.33	1.15	22.67	0.58	115.33	3.06	267.33	7.02
T4 (0.158)	6.00	1.00	13.33	1.15	23.33	2.08	118.33	2.52	278.00	11.14
T5 (0.501)	6.33	0.58	13.00	2.65	22.00	2.00	118.67	4.16	272.00	6.00
PC	169.33	18.04	502.67	14.05	1346.67	34.02	1662.67	42.02	1456.00	52.46

 

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)	4.00	0.00	10.67	0.58	19.67	1.53	104.00	2.00	213.33	4.16
VC (0.00)	6.67	0.58	14.00	2.00	22.33	1.53	117.33	3.06	282.67	3.06
T1 (0.005)	4.67	0.58	11.33	1.15	20.67	1.53	104.00	2.00	244.67	9.87
T2 (0.0.016)	5.00	0.00	11.00	1.00	20.00	2.00	114.33	1.53	243.33	11.72
T3 (0.050)	5.67	0.58	12.67	1.15	21.33	2.08	110.67	1.53	263.33	9.45
T4 (0.158)	6.33	0.58	12.33	0.58	20.33	2.08	109.00	9.17	260.67	7.57
T5 (0.501)	5.33	0.58	13.33	1.15	20.67	2.08	113.67	7.09	277.33	4.62
PC	156.00	16.00	1122.67	36.07	913.33	41.63	1520.00	72.00	1509.33	33.31

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-INCUBATIONMETHOD
(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)	4.67	0.58	11.67	1.53	19.33	1.15	111.33	3.06	241.33	11.02
VC (0.00)	7.00	1.00	15.00	1.00	24.33	0.58	128.00	4.00	269.33	2.31
T1 (0.005)	5.00	1.00	12.33	1.53	20.67	1.53	116.00	4.00	242.67	6.43
T2 (0.0.016)	4.33	0.58	13.00	1.73	20.00	2.00	118.67	5.03	242.00	2.00
T3 (0.050)	6.33	0.58	14.00	1.00	21.67	1.53	122.00	4.00	253.33	3.06
T4 (0.158)	6.00	1.00	13.67	0.58	20.33	2.52	116.67	3.06	244.00	4.00
T5 (0.501)	6.67	0.58	14.00	1.00	23.67	1.53	124.00	2.00	262.00	8.72
PC	180.00	14.00	400.00	28.00	1234.67	54.45	1514.67	40.27	1512.00	40.00

 

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	11.67	1.53	20.00	1.00	112.00	2.00	240.00	6.00
VC (0.00)	6.67	0.58	14.67	0.58	24.00	2.00	125.33	3.06	267.33	3.06
T1 (0.005)	4.67	0.58	12.00	1.00	20.67	0.58	117.33	3.06	241.33	3.06
T2 (0.0.016)	6.00	1.00	12.33	0.58	22.00	1.73	112.67	1.15	242.00	10.39
T3 (0.050)	6.33	0.58	12.67	0.58	21.33	1.53	120.00	2.00	252.67	7.57
T4 (0.158)	6.00	1.00	12.33	1.53	21.00	2.65	119.67	1.53	244.67	3.06
T5 (0.501)	6.67	0.58	13.67	1.53	23.33	0.58	120.67	3.06	257.33	9.45
PC	194.00	7.21	1234.67	64.17	834.67	82.40	1202.67	53.27	1597.33	39.46

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

Applicant's summary and conclusion

Conclusions:

The test chemical 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:

This study was performed to investigate the potential of with Methyl 5-nitrohydrogen.isophthalate (CAS No: 1955-46-0) 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 theSalmonella typhimuriumstrains 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., 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

(The test item concentration values have been incorporated up to three decimal places in the report).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Methyl 5-nitrohydrogen.isophthalate (CAS No: 1955-46-0) 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 positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item with Methyl 5-nitrohydrogen.isophthalate (CAS No: 1955-46-0)did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.